Chemically modified guide rnas for genome editing with cas9

ABSTRACT

Provided herein are compositions for gene modification or editing and methods of using same to treat or prevent certain conditions. Specific compositions and methods capable of safely and effectively editing gene targets expressed in the liver to durably lower LDL-C thereby treating a leading cause of cardiovascular disease are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119 from ProvisionalApplication Ser. No. 63/007,803, filed Apr. 9, 2020; ProvisionalApplication Ser. No. 63/007,797, filed Apr. 9, 2020; ProvisionalApplication Ser. No. 63/136,087, filed Jan. 11, 2021; ProvisionalApplication Ser. No. 63/045,032, filed Jun. 26, 2020; ProvisionalApplication Ser. No. 63/045,033, filed Jun. 26, 2020, the disclosures ofwhich are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 8, 2021, isnamed 53989-708_601_SL.txt and is 1,105,762 bytes in size.

FIELD OF DISCLOSURE

Provided herein are compositions for gene modification or editing, andmethods of using same that are capable of treating or preventing certainconditions, such as cardiovascular disease and conditions or diseasesassociated therewith such as diabetes.

BACKGROUND

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by references to the same extentas if each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.It is not an admission that any publication or information specificallyor implicitly referenced herein is prior art or necessarily relevant tothe claimed subject matter. To the extent publications or patents orpatent applications incorporated by reference contradict or areinconsistent with the disclosure contained in the specification, suchcited or incorporated references should be considered supplementary tothis disclosure with the understanding that the specification isintended to supersede and/or take precedence over any irreconcilableinconsistencies or contradictory material.

SUMMARY

The inventive subject matter disclosed in this application is directedto compositions capable of editing a polynucleotide or target gene andmethods of use of those compositions. The compositions and theconstituent components thereof, individiualy and in combination, areconsidered separate aspects of the inventive subject matter, which maybe defined and claimed by their respective physical and/or functionalattributes described herein in any combination without limitation. Thebreadth, specificity, and variation of the inventive subject matter arefurther illustrated by specific aspects summarized here.

One significant aspect of the inventive subject matter described hereinis directed to the treatment of cardiovascular disease (CVD), which isthe leading cause of death worldwide, responsible for nearly one inthree deaths according to the World Health Organization. CVD is also aleading contributor to reductions in life expectancy and is one of themost expensive health conditions to care for. According to the Centersfor Disease Control and Prevention (CDC), CVD is a significant economicburden, costing the U.S. healthcare system more than $320 billion peryear in annual costs and lost productivity. CVD collectively refers todiseases of the heart and blood vessels, which are diagnosed as eitheratherosclerotic cardiovascular disease (ASCVD) or cardiomyopathy, amongothers. ASCVD is a large subset of CVD for which cholesterol drives thedevelopment of atherosclerotic plaque, a mixture of cholesterol, cellsand cellular debris in the wall of a blood vessel that results in thehardening of the arteries. The current cornerstone of the treatment andprevention of ASCVD is to lower cumulative exposure to blood lipidsaiming to maintain low-density lipoprotein cholesterol (LDL-C, commonlyknown as “bad” cholesterol) and/or triglycerides as low as possible foras long as possible. There is significant evidence, for example,demonstrating that individuals who maintain LDL-C at sufficiently lowlevels over a sufficiently long period of time are substantially lesslikely to develop ASCVD. The relationship between the lowering of LDL-Cand reduction in ASCVD is amongst the best understood of allrelationships in medicine. It has been shown that lowering LDL-C by 39mg/dL for five years in a patient with established ASCVD reduces ASCVDrisk by 21%, whereas that same 39 mg/dL degree of LDL-C reduction over alifetime reduces risk of a first ASCVD event by 88%. The currentstandard of care is a chronic care model that typically requiresnumerous daily pills and/or intermittent injections and ofteninsufficiently controls cumulative exposure to LDL-C. Despite theavailability of such chronic care therapies, cumulative exposure toLDL-C is often insufficiently controlled in many patients with ASCVD,and a large fraction of individuals with established ASCVD have LDL-Clevels above the recommend goal. Higher cumulative LDL-C exposure leadsto accelerated cholesterol plaque build-up in the heart or neck arteriesand the rupture of which can result in heart attack, cardiac death,stroke and the need for invasive medical procedures, such asintracoronary stenting and coronary artery bypass surgery.

One aspect, disclosed herein, are compositions and methods that arecapable of safely and effectively editing gene targets expressed in theliver to durably lower LDL-C and/or triglycerides thereby treating CVDsuch as ASCVD.

Another aspect, disclosed herein, is the efficacy and safety of the geneediting compositions described herein when administered as asingle-course or dose (once-and-done) therapy, in repeat or successivedoses, and combination gene editing therapy doses. The efficacy andsafety of the compositions described herein are shown in in vitro and invivo studies described herein involving various cell and animalexperiments including mouse and non-human primate experiments, and theresults or performance of the compositions disclosed herein representedin those studies each constitute aspects of the invention.

Another aspect of the composition and methods disclosed herein relatesto the location where the edit in the gene is made, including forexample compositions and methods directed at editing a gene in thesplice site.

Another aspect of the compositions and methods disclosed herein are theconstituent guide RNAs (gRNAs) and base editors that comprise thecompositions that are capable of precisely editing a gene at a singlebase pair without imparting double-stranded breaks in the target gene.The compositions and method of use of those gRNAs and base editorsincluding nucleotide or mRNA sequences that express and encode the baseeditor constitute yet another aspect.

Another aspect, disclosed herein, are the lipid nanoparticle (LNPs)formulations that encapsulate the gRNA and base editor drug substances,the selection of an LNP, and the relative ratios between the variouscomponents of the drug substance compositions alone and as part of theLNP.

Another aspect, disclosed herein, is directed at the dosing of geneediting compositions, such as those described herein, and the impact ofdosing and repeat dosing on efficacy and safety profile indicia.

Another aspect, disclosed herein, are that the compositions and methodsof use include implementations that are designed to target or editspecific genes such PCSK9, ANGPTL3, APOC3, and/or Lp(a) and/or have animpact on the protein levels of proteins expressed by those genes.

In one aspect, provided herein is a single guide RNA that comprises: (a)a spacer sequence, wherein the spacer sequence comprises (i) one or morechemical modification(s) and (ii) one or more unmodified nucleotide(s)at select position(s), and (b) a tracr sequence having at least 70%identity to SEQ ID NO: 61, wherein the tracr sequence serve as a bindingscaffold for a Type II Cas protein, and wherein the tracr sequencecomprises (i) one or more chemical modification(s) and (ii) one or moreunmodified nucleotide(s) at select position(s). In some embodiments, thespacer sequence hybridizes with a target polynucleotide sequence in agene of interest when contacted with the target polynucleotide sequence,wherein the single guide RNA directs the Cas protein to effectalteration in the gene when administered to a mammalian subject. In someembodiments, the tracr sequence comprises unmodified nucleotides atpositions 2 to 7, 23 to 25, 27, 29, 31, 38, 39, 42 to 45 48, 49 and 62as numbered in SEQ ID NO: 61 or a corresponding position thereof. Insome embodiments, the tracr sequence comprises unmodified nucleotides atpositions 2 to 7, 13, 23 to 25, 27, 29, 31, 38, 39, 42 to 45, 48, 49, 53and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof.In some embodiments, the tracr sequence comprises unmodified nucleotidesat positions 2 to 7, 13, 23 to 25, 27, 29, 31, 38, 39, 42 to 45, 48, 49,53, 61, 68, 70 and 11 as numbered in SEQ ID NO: 61 or a correspondingposition thereof. In some embodiments, the tracr sequence comprisesmodified nucleotides at positions 1, 8 to 22, 26, 28, 30, 32 to 37, 40,41, 46, 47, 50 to 61, and 63 to 80 as numbered in SEQ ID NO: 61 or acorresponding position thereof. In some embodiments, the tracr sequencecomprises modified nucleotides at positions 1, 8 to 12, 14 to 22, 26,28, 30, 32 to 37, 40, 41, 46, 47, 50 to 52, 54 to 61, and 63 to 80 asnumbered in SEQ ID NO: 61 or a corresponding position thereof. In someembodiments, the tracr sequence comprises modified nucleotides atpositions 1, 8 to 12, 14 to 22, 26, 28, 30, 32 to 34, 37, 41, 46, 47, 50to 52, 54 to 60, 62 to 67, 69 and 72 to 80 as numbered in SEQ ID NO: 61or a corresponding position thereof. In some embodiments, more than 60%of the nucleotides in the tracr sequence with SEQ ID NO: 61 aremodified. In some embodiments, more than 70% of the nucleotides in thetracr sequence with SEQ ID NO: 61 are modified. In some embodiments, thetracr sequence comprises unmodified nucleotides at positions 13, 49, 53and 62 as numbered in SEQ ID NO: 61 or a corresponding position thereof.In some embodiments, the tracr sequence comprises unmodified nucleotidesat positions 49 and 62 as numbered in SEQ ID NO: 61 or a correspondingposition thereof. In some embodiments, the tracr sequence comprisesunmodified nucleotides at positions 13, 40, 49, 53, 61, 68, 70 and 71 asnumbered in SEQ ID NO: 61 or a corresponding position thereof. In someembodiments, the tracr sequence comprises unmodified nucleotides atpositions 13, 49 and 53 as numbered in SEQ ID NO: 61 or a correspondingposition thereof. In some embodiments, the tracr sequence comprisesmodified nucleotides at positions 1, 8, 21, 22, 26, 28, 30, 32 to 37,40, 41, 46 and 47 as numbered in SEQ ID NO: 61 or a correspondingposition thereof. In some embodiments, the tracr sequence comprises atleast 80% identity to SEQ ID NO: 61.In some embodiments, the tracrsequence comprises at least 90% identity to SEQ ID NO: 61. In someembodiments, the chemical modification comprises a 2′-OMe modification.In some embodiments, the chemical modification comprises a nebularin ora deoxynebularin. In some embodiments, the chemical modificationcomprises a phosphorothioate linkage. In some embodiments, the tracrsequence comprises SEQ ID No. 61, and the single guide RNA furthercomprises one or more phosphorothioate linkage at a 5′ end, at a 3′ end,at select internal positions or any combinations thereof. In someembodiments, the single guide RNA further comprises two and no more thantwo contiguous phosphorothioate linkages at the 5′ end, at the 3′ end orboth. In some embodiments, the single guide RNA further comprises threecontiguous phosphorothioate linkages at the 5′ end, at the 3′ end orboth. In some embodiments, the single guide RNA comprises the sequence5′-ususuNNN-3′ at the 3′ end, wherein N independently indicates aunmodified ribonucleotide, and wherein each u indicates2′-O-methyluridine and each s indicates phosphorothioate linkage. Insome embodiments, each N is uridine. In some embodiments, the singleguide RNA comprises the sequence 5′-ususuNNn-3′ at the 3′ end, whereineach N independently indicates a unmodified ribonucleotide, wherein then indicates a modified nucleotide, wherein each u indicates2′-O-methyluridine and wherein each s indicates phosphorothioatelinkage. In some embodiments, each N is uridine and the n is2′-O-methyluridine.

In another aspect, provided herein is a single guide RNA that comprises(i) a spacer sequence and (ii) a tracr sequence, wherein the spacersequence hybridizes with a target polynucleotide sequence in a PCSK9gene or an ANGPTL3 gene when contacted with the target polynucleotidesequence, wherein the tracr sequence binds a Type II Cas protein whencontacted with the Type II Cas protein, and wherein the single guide RNAcomprises a nebularine, a deoxynebularine, or a 2′-O-methylnebularine.In another aspect, provided herein is a single guide RNA that comprises(i) a spacer sequence and (ii) a tracr sequence, wherein the spacersequence hybridizes with a target polynucleotide sequence in a PCSK9gene or an ANGPTL3 gene when contacted with the target polynucleotidesequence, wherein the tracr sequence binds a Type II Cas protein whencontacted with the Type II Cas protein, and wherein the single guide RNAcomprises two and no more than two phosphorothioate linkages at a 5′ endor at a 3′ end. In some embodiments, the single guide RNA comprises twoand no more than two phosphorothioate linkages at the 5′ end and at the3′ end. In some embodiments, the single guide RNA comprises threephosphorothioate linkages at the 5′ end. In some embodiments, the singleguide RNA comprises three phosphorothioate linkages at the 3′ end. Insome embodiments, the two phosphorothioate linkages at the 5′ end aretwo contiguous phosphorothioate linkages at the first two nucleotidepositions of the 5′ end. In some embodiments, the two phosphorothioatelinkages at the 5′ end are within the first 3-10 nucleotides of the 5′end. In some embodiments, the two phosphorothioate linkages at the 3′end are two contiguous phosphorothioate linkages at the last twonucleotide positions of the 3′ end. In some embodiments, the twophosphorothioate linkages at the 3′ end are within the last 3-10nucleotides of the 3′ end. In some embodiments, the single guide RNA asprovided herein comprises the sequence 5′-UsUsUs-3′ at the 3′ end,wherein U indicates a uridine and s indicates a phosphorothioatelinkage. In some embodiments, the single guide RNA as provided hereincomprises the sequence 5′-UUU-3′ at the 3′end. In some embodiments, thetracr sequence binds the Type II Cas protein with increased bindingaffinity compared to a tracr sequence in an unmodified single guide RNA.In some embodiments, the Type II Cas protein is a Cas9 protein. In someembodiments, the Cas9 protein is a Streptococcus pyogenes Cas9.

In another aspect, provided herein is a single guide RNA that comprisesa guide RNA sequence selected from Table 1, wherein a, u, g, and cindicate 2′-OMe modified adenine, uridine, guanine, and cytidine,wherein s indicates a phosphorothioate linkage, wherein X indicates anebularine, wherein x indicates a 2′-O-methylnebularine and wherein dXindicates a 2′-deoxynebularine.

In another aspect, provided herein is a pharmaceutical composition forgene modification comprising the single guide RNA as provided herein anda Type II Cas protein or a nucleic acid sequence encoding the Type IICas protein. In some embodiments, the pharmaceutical composition asprovided herein further comprises a vector that comprises the nucleicacid sequence encoding the Type II Cas protein. In some embodiments, theType II Cas protein is a Cas9. In some embodiments, the pharmaceuticalcomposition as provided herein further comprises a pharmaceuticallyacceptable carrier.

In another aspect, provided herein is a lipid nanoparticle comprisingthe pharmaceutical composition as provided herein.

In another aspect, provided herein is a method for modifying a targetpolynucleotide sequence in a cell comprising introducing into the cellthe pharmaceutical composition as provided herein, wherein the singleguide RNA directs the Type II Cas protein to effect a modification inthe target polynucleotide sequence in the cell. In some embodiments, thetarget polynucleotide sequence is in a PCSK9 gene. In some embodiments,the modification reduces expression of functional PCSK9 protein encodedby the PCSK9 gene in the cell. In some embodiments, the targetpolynucleotide sequence is in an ANGPTL3 gene. In some embodiments, themodification reduces expression of functional ANGPTL3 protein encoded bythe ANGPTL3 gene in the cell. In some embodiments, the introduction isperformed via a lipid nanoparticle that comprises the composition.

In another aspect, provided herein is a method for treating orpreventing a condition in a subject in need thereof, the methodcomprising administering to the subject the pharmaceutical compositionas provided herein or the lipid nanoparticle as provided herein, whereinthe single guide RNA directs the Type II Cas protein to effect amodification in a target polynucleotide sequence in a cell of thesubject, thereby treating or preventing the condition. In someembodiments, the target polynucleotide sequence is in a PCSK9 gene. Insome embodiments, the target polynucleotide sequence is in an ANGPTL3gene. In some embodiments, the modification reduces expression offunctional PCSK9 protein encoded by the PCSK9 gene in the subject. Insome embodiments, the modification reduces expression of functionalANGPTL3 protein encoded by the ANGPTL3 gene in the subject. In someembodiments, the condition is an atherosclerotic vascular disease. Insome embodiments, the condition is an atherosclerotic vascular disease,hypertriglyceridemia, or diabetes. In some embodiments, the subjectexhibits a reduced blood LDL cholesterol level, and/or a reduced bloodtriglycerides level as compared to before the administration.

BRIEF DESCRIPTION OF THE DRAWINGS

The features, principles, advantages, and illustrative embodiments,implementations, analysis, and examples of the subject matter of thisapplication are set forth herein including in the appended claims withaspects of which being illustrated in the accompanying drawings ofwhich:

FIGS. 1A-IC illustrate the modes of operation of Cas9, cytidine baseeditors (CBE), and adenine base editors (ABE) respectively, along withrelevant terminology used in this application including “protospacer”,“PAM”, “spacer” (Mali, P. et al 2013 Nat Methods 10, 957-963; Anzalone,A V. 2020 Nat Biotechnol 38, 824-844). FIG. 1D is a schematicrepresentation of how mcPCSK9 guide RNA (gRNA)-Tracr disclosed hereinwere designed. Shown is a ribonucleoprotein (RNP)-single guide RNA(sgRNA) alignment with stem-loop guide intramolecular interactions (W—Cbase pairing at the stem). The tracrRNA sequence of the gRNA serves as abinding scaffold for the cas protein. When designing a gRNA, loopnucleotides can be aligned with a protein and the interaction of base(H-bond), 2′-hydroxyl (2′-OH), 4′-oxygen (ring-oxygen) of the sugarmoiety, phosphate linkage of each nucleotide to amino acid side chainsof the protein can be considered in the design of a gRNA-Tracr. Spatialarrangement of nucleotide within RNP-steric interaction, room toaccommodate bulky substitution like 2′-O-Methyl (2′-OMe) andphosphorothioate (PS) can be also taken into consideration. FIG. 1Ddiscloses SEQ ID NOS 70-71, respectively, in order of appearance.

FIG. 2 are two tables that describe how SpCas9 crystal structures werechosen in connection with designing a gRNAs disclosed herein based onstructure. The top panel is a table summarizing different components ofCRISPR/Cas system with Protein Data Bank (PDB) IDs and the state. Thebottom panel shows the root mean square deviation (RMSD) values afteralignment between the protein chains of each structure. The RNP state(4ZT0) and pre-catalytic ternary complex (5F9R) are the most relevant todetermine contacts related to RNP formation and contacts related tocatalysis. Some rearrangement of the protein occurs between these twostates. Massive rearrangements may occur in the protein between the apostate and the RNP.

FIG. 3 depicts a gRNA secondary structure of a sgRNA based on the 4ZT0and 5F9R crystal structures, in which the sgRNA is bound to SpCas9either as an RNP alone or as part of a ternary complex with DNA.Secondary structure relationships are shown in the Leontis and Westhofnomenclature (RNA, 2001, 7, 499-512) (SEQ ID NO: 72).

FIG. 4 depicts a gRNA secondary structure with a PCSK9 spacer showingpredicted contacts based on RNP PDB 4ZT0 (sgRNA+SpCas9 RNP). Positions1-10 and positions 83-100 were not modeled in this crystal structure dueto a lack of clear electron density (SEQ ID NO: 73). Black circle withwhite letter labels: protein contact, light gray with black letter:steric clash if 2′-O-Me incorporated or distant contact, dark grey withblack letter: RNA contact. As a person of skill in the art wouldunderstand, the term “clash” as used herein refers tophysically-unlikely overlapping atomic volumes in a structure.Incorporation of a 2′O-Me modification in these situations wouldpotentially result in structural rearrangement(s) that could bedetrimental to RNP function. As a person of skill in the art wouldunderstand, the term “contact” as used herein means a stable,non-covalent interaction between two functional groups, e.g. a hydrogenbond.

FIG. 5 depicts a gRNA secondary structure with a PCSK9 spacer showingpredicted contacts based on PDB 5F9R (precatalytic ternary complex) (SEQID NO: 74). Black circle with white letter labels: protein contact,light gray with black letter: steric clash if 2′-O-Me incorporated ordistant contact, dark grey with black letter: RNA contact.

FIG. 6 depicts plausible positions of 2′-OMe substitutions determined bystructure-based designs (SEQ ID NO: 73). Black circle with white letterlabels: 2′-OMe and phosphorothioate substitution, light gray circle withblack letter labels: 2′-OMe substitution only, white circle with blackletter labels: unmodified nucleotide.

FIG. 7A depicts three patterns of 2′-OMe position identified in thetracr region of the single guide RNA from structure-guided incorporationof 2′-O-methylribosugar modification that produced robust editing invivo: (1) mice: FIG. 16 , GA054 (tracr1) and GA055 (tracr2) and (2) NHP:FIG. 28 , GA096 (tracr1), GA097 (tracr2) and GA346 (tracr3). Blackcircle with white letter labels: 2′-OMe and phosphorothioatesubstitution, light gray circle with black letter labels: 2′-OMesubstitution only, white circle with black letter labels: unmodifiednucleotide. (SEQ ID NO: 73) FIG. 7B depicts the sequence alignment ofunmodified SEQ ID NO: 61, a reference tracrRNA sequence (“Lit Tracr”),tracr1, tracr2, and tracr3. (SEQ ID NO: 73)

FIG. 8 shows base editing of the target splice site by an adenosine baseeditor system in modifying PCSK9 in primary human hepatocytes. The darkline represents the percent splice site editing obtained using gRNAidentified a GA066.

FIG. 9 depicts a Sanger sequencing chromatogram demonstrating editing ofadenine base in the antisense strand at the splice donor at the end ofPCSK9 exon 1 (PCR amplification from the genomic DNA of the cellstransfected with the 2500-ng/mL dose), portraying how splice-sitedisruption results in an in-frame stop codon. Heterozygosity for anaturally occurring single nucleotide polymorphism (SNP) is evidentdownstream of the editing site. The scheme shows A-to-G base editing byan adenosine base editor system to knock-out PCSK9 in primary humanhepatocytes. FIG. 9 discloses SEQ ID NO: 75.

FIG. 10 depicts a Sanger sequencing chromatogram demonstrating editingof adenine base in the antisense strand at the splice donor at the endof ANGPTL3 exon 6 (PCR amplification from the genomic DNA of the cellstransfected with the 2500-ng/mL dose), portraying how splice-sitedisruption results in an in-frame stop codon. A-to-G base editingeffected by an adenosine base editor system in modifying ANGPTL3 inprimary human hepatocytes. FIG. 10 discloses SEQ ID NO: 76.

FIG. 11 shows three sanger sequencing chromatograms from PCR amplifiedgenomic DNA isolated from: 1) untreated primary hepatocytes (top panel);2) SpCas9 mRNA/PCSK9 gRNA GA097 treated primary hepatocytes (middlepanel); 3) ABE8.8 mRNA/PCSK9 gRNA treated primary hepatocytes (bottompanel). The PCSK9-gRNA GA097 protospacer sequence is highlighted ingrey. The arrow points to the position 6 of the protospacer that istargeted for A-to-G base editing by the ABE8.8 editor. The scissorsdepict the general site of double stranded break that occurs uponcutting by the SpCas9 nuclease at this target site. SpCas9 mRNA andGA097 delivery in cells resulted in significant gene editing at the siteof the double strand break (as denoted with the scissors) but this wasnot seen in the ABE8.8 and GA097 delivery in cells. Instead, the samegRNA GA097 in combination with ABE8.8 mRNA resulted in robust A-to-Gbase editing. FIG. 11 discloses SEQ ID NO 77-79, respectively in orderof appearance.

FIG. 12 depicts a schematic showing potential splicing outcomes withdisruption of splice donor or splice acceptor sequences. Other outcomesare possible, such as inclusion of part of intron 1 in the splicingproduct. Alteration of the splice donor or splice acceptor sites areshown (top panel). Alternative splice donor sites within PCSK9 intron 1resulting from editing of PCSK9 exon 1 splice-donor adenine base inprimary human hepatocytes is shown in the bottom panel. *Four differentprimer pairs (Table 9) were used for RT-PCR of the control/treatedsamples.

FIG. 13 shows lack of guide RNA-dependent DNA off-target editing inprimary human hepatocytes. Human primary hepatocytes were incubated withgRNA (GA097 or GA346) and ABE8.8 mRNA as described in Example 4. Theoff-target reading was calculated as net adenine editing (proportion ofsequencing reads with alteration of one or more adenine bases inLNP-treated cells versus untreated cells) at the on-target PCSK9 siteand more than 50 candidate off-target PCSK9 sites in primary humanhepatocytes from four individual donors.

FIG. 14 depicts guide RNA-independent RNA editing, assessed inSpCas9-treated or ABE8.8-treated hepatocytes after 2 days (n=4biological replicates). Each replicate was compared against each of fouruntreated hepatocyte samples to eliminate any positions with editingthat were common to both conditions. The jitter plots portraytranscriptomic loci with editing in the treated sample (number indicatestotal edited loci identified in the treated sample, boxplot indicatesmedian ±interquartile range of proportion of edited reads across alledited loci in the sample). gRNA GA097, SpCas9 mRNA MS010, and ABE8.8mRNA MA004 were used in this study as described in example 4.

FIG. 15 shows PCSK9 and ANGPTL3 base editing with lipid nanoparticles(LNPs) formulated with an embodiment of base editor system, ABE mRNA anda guide RNA, in primary human hepatocytes and primary non-human primates(NHP) hepatocytes. The human-specific ANGPTL3 guide RNA showed lowediting efficiency in NHP hepatocytes within the concentrationevaluated, whereas the PCSK9 guide RNA cross-reactive to both human andNHP showed high editing efficiency in both cell lines.

FIG. 16 shows gene editing of Pcsk9 in mice (n=2-5 mice) via LNPscontaining SpCas9 mRNA MS002 (TriLink Biotechnologies) and mousePcsk9-targeting gRNA with different tracr designs (GA052, GA053, GA054,and GA055) at a 1:1 weight ratio. Wild-type C57BL/6 mice were dosed with2 mg/kg of the LNP test article. The mg/kg dose was calculated based ontotal RNA and the total RNA is the quantified sum of mRNA and gRNApresent in the LNP after formulation. Seven days after dosing, the micewere euthanized and genomic DNA was harvested from mouse liver, and thenassessed for base editing of the target site with next-generationsequencing. All four guide RNAs evaluated carry same spacer and samepattern of chemical modification within the first 20 nucleotides fromthe 5′-end, but all four differ in chemical modification pattern withinthe tracr between nucleotides at position 21 and at position 100 of the100-mer guide RNA.

FIG. 17 shows base editing of PCSK9 in mice (n=5) via LNPs with anembodiment of base editor system, ABE mRNA MA004 and PCSK9 guide RNAGA256 (targeting mouse intron 1 splice donor). Wild-type C57BL/6 micewere dosed with 2 mg/kg total RNA of the LNP test article. Seven daysafter dosing, the mice were euthanized and genomic DNA was harvestedfrom mouse liver, and then assessed for base editing of the target sitewith next-generation sequencing.

FIG. 18 depicts editing of the Pcsk9 exon 1 splice-donor adenine base inwild-type mouse liver, assessed 1 week following treatment withdifferent doses of same LNP formulation with ABE8.8 mRNA MA004 and Pcsk9gRNA GA256 (n=4 to 5 mice per dosing group, bar indicates mean editingin group).

FIG. 19 depicts editing of the Pcsk9 exon 1 splice-donor adenine base inwild-type mouse liver, after dosed with LNPs at 0.05 mg/kg total RNAdose containing different ratios of gRNA GA256 and mRNA MA002.Additional guides GA255 and GA257 with different chemical modificationswere also assessed for base editing efficiency, at mRNA to gRNA 1:1 wtratio.

FIG. 20 shows the results from dosing LNPs containing ABE8.8 mRNA andmouse Angptl3-targeting gRNAs (GA258, GA259, GA260, GA349, GA353) at0.05 mg/kg total RNA dose at a 1:1 weight ratio into mice. GA258, GA259and GA260 contain three different structure-guided tracr design. GA349and GA353 tracr designs were from published literature (Cell Reports,2018 22, 2227-2235). The mice were later euthanized and genomic DNA washarvested from mouse liver, and then assessed for base editing of thetarget splice site with next-generation sequencing.

FIG. 21 is a schematic showing the general dosing strategy forintroducing adenine base editing of a target gene in NHP via LNPsformulated with an embodiment of base editor system, ABE mRNA and aguide RNA, and subsequent analysis after 2 weeks.

FIG. 22 shows that administration of LNPs formulated with an embodimentof base editor system, ABE mRNA MA002 and PCSK9 guide RNA GA066, at 1mg/kg and 3 mg/kg total RNA dose to cynomolgus monkeys via intravenousinfusion, induced adenine base editing at the PCSK9 target splice sitein the liver of cynomolgus monkeys.

FIG. 23 shows that administration of LNPs formulated with an embodimentof base editor system, ABE mRNA MA004 and PCSK9 guide RNA GA066, tocynomolgus monkeys via intravenous infusion resulted in reduction in theblood PCSK9 protein level compared to pre-dosing levels at 2 weeks afterdosing.

FIG. 24 shows that administration of LNPs at 3 mg/kg formulated with anembodiment of base editor system, ABE mRNA MA002 and PCSK9 guide RNAGA066, to cynomolgus monkeys via intravenous infusion resulted inreduction of the blood low-density lipoprotein cholesterol (LDL-C) levelat 1 and 2 weeks after dosing compared to pre-dosing levels.

FIGS. 25A-25C show short-term adenine base editing of PCSK9 in non-humanprimates. FIG. 25A depicts editing of the PCSK9 exon 1 splice-donoradenine base in the livers of cynomolgus monkeys receiving anintravenous infusion of a 1 mg/kg dose of an LNP formulation with ABE8.8mRNA MA004 and PCSK9 gRNA GA097 with necropsy at either 2 weeks (threeanimals) or 24 hours (two animals) following treatment. For each animal,editing was assessed in samples collected from sites distributedthroughout the liver (n=8 samples; bar indicates mean editing inanimal). Reduction of the blood PCSK9 protein level (FIG. 25B) or bloodLDL-C level (FIG. 25C) in the three animals that underwent necropsy at 2weeks following treatment are shown, comparing the level at 2 weeksversus the baseline pre-treatment level (n=1 blood sample per animal).

FIGS. 26A-26C shows adenine base editing of PCSK9 in non-human primates.FIG. 26A depicts editing of the PCSK9 exon 1 splice-donor adenine basein the livers of cynomolgus monkeys receiving an intravenous infusion of0.5, 1.0, or 1.5 mg/kg dose of an LNP formulation with ABE8.8 mRNA MA004and PCSK9 gRNA GA346. Reduction of the blood PCSK9 protein level (FIG.26B) or blood LDL-C level (FIG. 26C) for the animals are shown.

FIG. 27 depicts tissue distribution of editing of the PCSK9 exon 1splice donor adenine base in the three animals that underwent necropsyat 2 weeks following treatment (n=1 sample per animal for each indicatedorgan except liver; the liver data represent the means shown in acalculated from eight liver samples each). The LNP constituted with ABEmRNA MA004 and guide RNA GA346 was used in this study, and the doseadministered was 0.5 mg/kg.

FIG. 28 illustrates editing of the PCSK9 exon 1 splice-donor adeninebase editing in the livers of cynomolgus monkeys following intravenousinfusions of individual lipid nanoparticles (LNPs) constituted with ABEmRNA MA004 and different guide RNAs with same spacer but with differenttracer modifications. The guide RNAs used in this study were GA066,GA096, GA097 and GA346. The gRNA GA097 from two different sources wereused in the same study and the sources are identified as (1) and (2).For tracr comparison studies the LNPS were dosed at 1 mg/kg total RNAdose where the guide RNA and mRNA were mixed at 1:1 weight ratio. TheGA066 with published tracr design (Cell Reports, 2018 22, 2227-2235)produced lower base editing in monkey compared to all other tracrdesigns (FIG. 7 )—GA095, GA097, and GA346—under same experimentalconditions.

FIG. 29 shows SpCas9 nuclease versus adenine base editing of PCSK9 innon-human primates. LNPs containing either SpCas9 mRNA and PCSK9 gRNA,(MS010/GA097) or ABE8.8 mRNA and PCSK9 gRNA (MA004/GA097) were infusedintravenously in cynomolgus monkeys. The MS010/GA097 LNP was dosed at0.75 and 1.5 mg/kg, and the MA004/GA097 LNP was dosed at 1 mg/kg totalRNA dose. The MS010/GA097 LNP test article at 1.5 mg/kg produced lowsingle digit gene editing in NHP whereas the ABE/GA097 test articleproduced about 40% adenine base editing at 1 mg/kg, showing therobustness of ABE base editor over SpCas9 system. All LNPs used in thisstudy were prepared using same excipients and compositions.

FIG. 30 shows adenine base editing of PCSK9 in non-human primates. Thisgraph depicts editing of the PCSK9 exon 1 splice-donor adenine base inthe livers of cynomolgus monkeys (each bar is an individual animal, withediting recorded for multiple sampling areas) receiving an intravenousinfusion of 0.5, 1.0, 1.5, or 3 mg/kg total RNA dose of LNP #1. LNP #2at 3 mg/kg total RNA was dosed as a benchmark from a previous study.Both LNP formulations contain ABE8.8 mRNA MA004 and PCSK9 gRNA GA346.

FIG. 31 shows the reduction of PCSK9 protein levels from basal on Day15, from the same experiment described in FIG. 30 .

FIG. 32 depicts editing of the PCSK9 exon 1 splice-donor adenine base inthe livers of four cynomolgus monkeys received an intravenous infusionof a 3 mg/kg total RNA dose of an LNP formulation with ABE8.8 mRNA MA004and PCSK9 gRNA GA066. For each animal, editing was assessed in a liverbiopsy sample at 2 weeks following treatment (n=1 sample per animal).

FIG. 33 depicts reduction of the blood PCSK9 protein levels in the fouranimals from FIG. 32 (animals that received 3 mg/kg total RNA dose of anLNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA066) and in twocontemporaneous control animals that received phosphate-buffered saline,comparing levels at various time points following treatment versus thebaseline pre-treatment level (mean±standard deviation for each group,n=4 or n=2, at each time point). The dotted lines indicate 100% and 10%of baseline levels, respectively.

FIG. 34 depicts reduction of the blood LDL-C level in the four animalsfrom FIG. 32 (animals that received 3 mg/kg total RNA dose of an LNPformulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA066) and in twocontemporaneous control animals that received phosphate-buffered saline,comparing levels at various time points following treatment versus thebaseline pre-treatment level (mean±standard deviation for each group,n=4 or n=2, at each time point). The dotted lines indicate 100% and 40%of baseline levels, respectively (top panel). The absolute values ofindividual animals are shown in the bottom panel.

FIG. 35 depicts reduction of Lipoprotein(a) in the four animals fromFIG. 32 and in two contemporaneous control animals that receivedphosphate-buffered saline, comparing levels at various time pointsfollowing treatment versus the baseline pre-treatment level(mean±standard deviation for each group, n=4 or n=2, at each timepoint).

FIG. 36 shows long-term phenotypic effects of liver PCSK9 base editingin non-human primates. Absolute values of aspartate aminotransferase(AST) (top panel), and alanine aminotransferase (ALT) (bottom panel) inthe individual animals portrayed in FIG. 32 (n=4 animals treated with 3mg/kg dose of an LNP formulation with ABE8.8 mRNA and PCSK9-gRNA, andn=2 animals treated with phosphate-buffered saline) at various timepoints following treatment.

FIGS. 37A-37G show liver function markers of individual animals. AST(FIG. 37A, FIG. 37B), ALT (FIG. 37A, FIG. 37C), Alkaline phosphate (FIG.37A, FIG. 37D), gamma-glutamyltransferase (FIG. 37A, FIG. 37E), totalbilirubin (FIG. 37A, FIG. 37F), and albumin (FIG. 37A, FIG. 37G), up to15 days post dose, from cynomolgus monkeys that received an intravenousinfusion of a 0.5, 1.0, or 1.5 mg/kg dose of an LNP formulation withABE8.8 mRNA and PCSK9 gRNA.

FIG. 38 is a schematic of the representative candidate ONE-seq sitesusing a specific library designed against the cynomolgus genome. ThePCSK9 protospacer is depicted at the top with a ONE-seq score of 1.00.All sites listed are ranked by decreasing ONE-seq score, with mismatchesto the protospacer sequence identified. FIG. 38 discloses SEQ ID NO: 80,2193-2196, 566, 2197-2205, 675, 2206-2220, 564, 2221-224, 638, 644, 506,621, 2225-2226, 681, and 2227-2230, respectively, in order ofappearance.

FIG. 39 shows gRNA-dependent, DNA off-target analysis of the sitesidentified in FIG. 38 . Samples from cynomolgus primary hepatocytes (toppanel) or cynomolgus monkey livers (bottom panel) were assessed for netA>G base editing (n=3 treated, n=3 untreated samples).

FIG. 40 shows net A>G % base editing at one off-target site (C5),identified in FIG. 39 , from livers of NHPs that received either 0.5,1.0, or 1.5 mg/kg LNP.

FIG. 41 shows base editing of ANGPTL3 in NHPs via LNPs formulated withan embodiment of base editor system, ABE mRNA MA004 and ANGPTL3 guideRNA GA067. Liver editing (left panel), ANGPTL3 protein levels (middlepanel), and triglyceride levels (right panel) are shown for three NHPs.

FIG. 42 shows simultaneous ANGPTL3 and PCSK9 base editing with lipidnanoparticles (LNPs) formulated with an embodiment of base editorsystem, ABE mRNA MA002 and dual guide RNAs GA095 (hcPCSK9) and GA098(hANGPTL3), in human primary hepatocytes at concentrations ranging from0-2500 ng/test article/mL.

FIG. 43 shows Day 15 and Day 44 liver biopsy adenosine base editingresults. NHPs were dosed with LNPs formulated with ABE8.8 mRNA MA004 andeither a gRNA targeting PCSK9 (GA346) or a gRNA targeting ANGPTL3(GA347) via intravenous infusion at a total RNA doses ranging from 0.5-2mg/kg. Two weeks after administration of test article, biopsies wereperformed to assess base editing. After 30 days from the initiation ofthe study, the opposite LNP was administered. Following a second biopsyafter an additional 2 weeks, the gDNA was extracted, and base editingwas assessed using next generation sequencing. Results for PCSK9 baseediting (top panel) and ANGPTL3 base editing (bottom panel) are shown. 2mg/kg total RNA dose of LNP encapsulating ABE8.8 mRNA, PCSK9 gRNA GA346and ANGPTL3 gRNA GA347 at 1:0.5:0.5 weight ratio produced robustsynchronized PCSK9 and ANGPTL3 gene editing (Example 10).

FIG. 44 illustrates the corresponding % change in PCSK9 (top panel) andANGPTL3 (bottom panel) protein levels from NHPs described in FIG. 43 .

FIG. 45 illustrates that repeat LNP dosing in NHPs causes additiveadenosine base editing in the liver, post Day 14, Day 46, and Day75liver biopsies. NHPs were dosed with either LNP #1 or LNP #2 formulatedwith ABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097) viaintravenous infusion at a total RNA doses of 0.5 mg/kg (see Example 10for details on dosing intervals and related details).

FIG. 46 illustrates that repeat LNP dosing in NHPs causes additive baseediting in the liver and translates to dose-dependent additive decreasein plasma PCSK9 protein levels over 90 days. As described in FIG. 45 ,NHPs were repeat dosed with LNPs formulated with ABE8.8 mRNA MA004 and agRNA targeting PCSK9 (GA097). NHPs were dosed via intravenous infusionat a total RNA doses of 0.5 mg/kg at days 0, 30, and 60 (arrow isillustrated on graph to depict dosing). For description of analysis ofPCSK9 protein levels, see detailed methods section.

FIG. 47 illustrates that repeat LNP dosing in NHPs causes additive baseediting in the liver with only transient liver marker increase, and thetransient liver marker increase correlates well with the day of eachdose administered. The data shows 71 days of the liver marker levels ofALT, AST, total bilirubin, and creatine kinase post first dose (seeExample 10, FIG. 45 for details).

FIG. 48 illustrates that repeat LNP dosing in NHPs causes additive baseediting in the liver, with only transient liver marker increase showingup to 71 days post dose of the liver enzyme levels of LDH, GLDH, GGT,and ALP, in NHPs (See Example 10, FIG. 45 for details).

FIGS. 49A and 49B illustrate that base editing of ANGPTL3 results inlong-term decreased ANGPTL3 protein and triglyceride levels after asingle dose of LNP constituted with ABE8.8 mRNA MA004 and ANGPTL3 gRNAGA067. The results illustrates the effect of long-term adenine baseediting of ANGPTL3 on ANGPTL3 protein (FIG. 49A) and triglycerides (FIG.49B) in non-human primates over 6 months. ANGPTL3 protein (96%reduction) and triglyceride levels were substantially decreased uponsingle dose administration of the LNP, and remain stably reduced formore than 170 days (see Example 10 for details).

FIGS. 50A-50E illustrate the effect of LNP dosing in NHPs on cytokineactivation and immune response. Cynomolgus monkeys received intravenousinfusions of 0.5 mg/kg doses at specified time points (FIG. 50A and FIG.50B) of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9 gRNA GA346.Blood was collected at timepoints specified and the graph, and IP-10 andMCP-1 were analyzed. In additional studies, IL-6, MCP-1, and SC5b-9(FIG. 50C, FIG. 50D, and FIG. 50E, respectively) were analyzed atdifferent time points from blood collected from NHPs that received anintravenous infusion of 1.0 mg/kg total RNA dose of LNP formulated withMA004 and PCSK9 gRNA GA346.

FIG. 51 illustrates liver editing in NHPs at 15 days after treatmentwith an LNP containing SpCas9 mRNA/gRNA. FIG. 51 shows the results fromgene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgusmonkeys received an intravenous infusion of a 1.5 mg/kg dose of an LNPformulation with SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3(GA261-GA263) or PCSK9 (GA266-GA271). Upon necropsy after 2 weeks, twopieces from each liver lobe (8 pieces total) were isolated and gDNA wasextracted. Samples were processed as described in the detailed methodssection. Indel % was analyzed for each separate piece and are graphed asindividual points. High editing efficiency was observed in most NHPlivers.

FIG. 52 illustrates LDL-C levels in NHPs at 15 days after treatment withSpCas9/gRNA. FIG. 52 shows the reduction of LDL-C from gene editing ofANGPTL3 or PCSK9 in non-human primates. Cynomolgus monkeys received anintravenous infusion of a 1.5 mg/kg dose of an LNP formulation withSpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3 (GA261-GA263) orPCSK9 (GA266-GA271. Samples were processed as described in the detailedmethods section. All NHPs that received LNPs with SpCas9 mRNA/PCSK9 gRNAhad at least 35% reduction in circulating LDL-C levels. Although moremodest, LNPs with SpCas9 mRNA/ANGPTL3 gRNA had 10-25% reduction incirculating LDL-C levels.

FIG. 53 illustrates triglyceride levels in NHPs at 15 days aftertreatment with SpCas9/gRNA. FIG. 53 shows the triglyceride levels fromgene editing of ANGPTL3 or PCSK9 in non-human primates. Cynomolgusmonkeys received an intravenous infusion of a 1.5 mg/kg dose of an LNPformulation with SpCas9 mRNA MS004 and one gRNA targeting either ANGPTL3(GA261-GA263) or PCSK9 (GA266-GA271. Samples were processed as describedin the detailed methods section. NHPs that received LNPs with SpCas9mRNA/ANGPTL3 gRNA had around 10-50% reduction in triglyceride levels.NHPs that received LNPs with SpCas9 mRNA/PCSK9 gRNA did not show asignificant reduction in triglyceride levels.

FIGS. 54A and 54B illustrate that PACE-modifications to the gRNAdecrease off-target editing efficiency. Human primary hepatocytes weretransfected at 2500, 1250, 500, and 250 ng/test article/mL with SpCas9mRNA (commercially purchased from Trilink) and a gRNA targeting PCSK9with modifications to the tracr. Genomic DNA was processed, sequenced,and analyzed as described in the detailed methods section. GA156 wastransfected to serve as a positive control. GA248 and GA249 containPACE-modifications to the gRNA that have previously been demonstrated todecrease off-target editing efficiency. Indeed, although GA248 and GA249had lower on-target editing compared to the unmodified gRNA, GA156 (FIG.54A), GA248 and GA249 showed decreased off-target editing at anidentified off-target site (FIG. 54B).

FIG. 55A illustrates the GC comparison of ABE-encoding nucleotides,MA004, MA019, MA020, MA021, and ABE8.8 m (Table 23), as well as a moredetailed look at MA004 (FIG. 55A, bottom panel). FIG. 55B illustratesthe editing % obtained using ABE-encoding nucleotides, MA004, MA019,MA020, and MA021 (Table 23).

DETAILED DESCRIPTION

Certain specific details of this description are set forth in order toprovide a thorough understanding of various embodiments. However, oneskilled in the art will understand that the present disclosure may bepracticed without these details. In other instances, well-knownstructures have not been shown or described in detail to avoidunnecessarily obscuring descriptions of the embodiments.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure, suitable methods, andmaterials are described below. Further, headings provided herein are forconvenience only and do not interpret the scope or meaning of theclaimed disclosure. It is contemplated that any embodiment discussed inthis specification can be implemented with respect to any method orcomposition of the present disclosure, and vice versa. Furthermore,compositions of the present disclosure can be used to achieve methods ofthe present disclosure.

Definitions

To facilitate an understanding of the present disclosure, a number ofterms and phrases are defined below.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise.

It should also be noted that the term “or” is generally employed in itssense including “and/or” unless the content clearly dictates otherwise.The terms “and/or” and “any combination thereof” and their grammaticalequivalents as used herein, can be used interchangeably. These terms canconvey that any combination is specifically contemplated. Solely forillustrative purposes, the following phrases “A, B, and/or C” or “A, B,C, or any combination thereof” can mean “A individually; B individually;C individually; A and B; B and C; A and C; and A, B, and C.” The term“or” can be used conjunctively or disjunctively, unless the contextspecifically refers to a disjunctive use.

The term “about” or “approximately” can mean within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, within5-fold, and more preferably within 2-fold, of a value. Where particularvalues are described in the application and claims, unless otherwisestated the term “about” meaning within an acceptable error range for theparticular value should be assumed.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

As used herein, “some embodiments,” “an embodiment,” “one embodiment,”“embodiments” or “other embodiments” means that a particular feature,structure, or characteristic described in connection with theembodiments is included in at least some embodiments, but notnecessarily all embodiments, of the present disclosures.

The term “nucleic acid” as used herein refers to a polymer containing atleast two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) ineither single- or double-stranded form and includes DNA and RNA.“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides. A nucleic acid includes anyoligonucleotide or polynucleotide, with fragments containing up to 60nucleotides generally termed oligonucleotides, and longer fragmentstermed polynucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. DNA may be in the form of, e.g., antisense molecules, plasmidDNA, pre-condensed DNA, a PCR product, vectors, expression cassettes,chimeric sequences, chromosomal DNA, or derivatives and combinations ofthese groups. A ribooligonucleotide consists of a similar repeatingstructure where the 5-carbon sugar is ribose. Accordingly, the terms“polynucleotide” and “oligonucleotide” can refer to a polymer oroligomer of nucleotide or nucleoside monomers consisting ofnaturally-occurring bases, sugars and intersugar (backbone) linkages.Additionally, nucleic acids include nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, nonstandard, and/or non-naturally occurring, and which havesimilar binding properties as the reference nucleic acid. The nucleicacid may be modified at the base moiety (e.g., at one or more atoms thattypically are available to form a hydrogen bond with a complementarynucleotide and/or at one or more atoms that are not typically capable offorming a hydrogen bond with a complementary nucleotide), sugar moiety,or phosphate backbone. Backbone modifications can include, but are notlimited to, a phosphorothioate, a phosphorodithioate, aphosphoroselenoate, a phosphorodiselenoate, a phosphoroanilothioate, aphosphoraniladate, a phosphoramidate, and a phosphorodiamidate linkage.A phosphorothioate linkage substitutes a sulfur atom for a non-bridgingoxygen in the phosphate backbone and delays nuclease degradation ofoligonucleotides. A phosphorodiamidate linkage (N3′→P5′) allowspreventing nuclease recognition and degradation. Backbone modificationscan also include having peptide bonds instead of phosphorous in thebackbone structure (e.g., N-(2-aminoethyl)-glycine units linked bypeptide bonds in a peptide nucleic acid), or linking groups includingcarbamate, amides, and linear and cyclic hydrocarbon groups.Oligonucleotides with modified backbones are reviewed in Micklefield,Backbone modification of nucleic acids: synthesis, structure andtherapeutic applications, Curr. Med. Chem., 8 (10): 1157-79, 2001 andLyer et al., Modified oligonucleotides-synthesis, properties andapplications, Curr. Opin. Mol. Ther., 1 (3): 344-358, 1999. Nucleic acidmolecules described herein may contain a sugar moiety that comprisesribose or deoxyribose, as present in naturally occurring nucleotides, ora modified sugar moiety or sugar analog. The examples of modified sugarmoieties include, but are not limited to, 2′-O-methyl,2′-O-methoxyethyl, 2′-O-aminoethyl, 2′-Flouro, N3′→P5′ phosphoramidate,2′dimethylaminooxyethoxy, 2′ 2′dimethylaminoethoxyethoxy,2′-guanidinidium, 2′-O-guanidinium ethyl, carbamate modified sugars, andbicyclic modified sugars. 2′-O-methyl or 2′-O-methoxyethyl modificationspromote the A-form or RNA-like conformation in oligonucleotides,increase binding affinity to RNA, and have enhanced nuclease resistance.Modified sugar moieties can also include having an extra bridge bond(e.g., a methylene bridge joining the 2′-0 and 4′-C atoms of the ribosein a locked nucleic acid) or sugar analog such as a morpholine ring(e.g., as in a phosphorodiamidate morpholino). Examples of such analogsand/or modified residues include, but are not limited to diaminopurine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acidmethylester, 5-methyl-2-thiouracil, 3-(3-amino-3- N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, methyl phosphonates, chiral-methylphosphonates, 2′-O-methyl ribonucleotides, peptide-nucleic acids (PNAs),and the like. In some cases, nucleotides may include modifications intheir phosphate moieties, including modifications to a triphosphatemoiety. Non-limiting examples of such modifications include phosphatechains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8,9, 10 or more phosphate moieties) and modifications with thiol moieties(e.g., alpha-thiotriphosphate and beta-thiotriphosphates). Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of properties such as, for example, enhanced cellular uptake,reduced immunogenicity, and increased stability in the presence ofnucleases. Thus, the terms “polynucleotide” and “oligonucleotide” canalso include polymers or oligomers comprising non-naturally occurringmonomers, or portions thereof, which function similarly.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions), alleles, orthologs, SNPs, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)).

The present disclosure encompasses isolated or substantially purifiednucleic acid molecules and compositions containing those molecules. Asused herein, an “isolated” or “purified” DNA molecule or RNA molecule isa DNA molecule or RNA molecule that exists apart from its nativeenvironment. An isolated DNA molecule or RNA molecule may exist in apurified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or biologically active portion thereof,is substantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In oneembodiment, an “isolated” nucleic acid is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in some embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. In some examples, a vector is an expression vector that iscapable of directing the expression of nucleic acids to which they areoperatively linked. The term “operably linked,” as used herein, meansthat the nucleotide sequence of interest is linked to regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence. The term “regulatory sequence,” as used herein, includes, butis not limited to promoters, enhancers and other expression controlelements. Such regulatory sequences are well known in the art and aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Examples ofexpression vectors include, but are not limited to, plasmid vectors,viral vectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors.

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably and refer to a polymer of amino acid residueslinked via peptide bonds and which may be composed of two or morepolypeptide chains. The terms “polypeptide,” “protein,” and “peptide”refer to a polymer of at least two amino acid monomers joined togetherthrough amide bonds. An amino acid may be the L-optical isomer or theD-optical isomer. More specifically, the terms “polypeptide,” “protein,”and “peptide” refer to a molecule composed of two or more amino acids ina specific order; for example, the order as determined by the basesequence of nucleotides in the gene or RNA coding for the protein.Proteins are essential for the structure, function, and regulation ofthe body's cells, tissues, and organs, and each protein has uniquefunctions. Examples are hormones, enzymes, antibodies, and any fragmentsthereof. In some cases, a protein can be a portion of the protein, forexample, a domain, a subdomain, or a motif of the protein. In somecases, a protein can be a variant (or mutation) of the protein, whereinone or more amino acid residues are inserted into, deleted from, and/orsubstituted into the naturally occurring (or at least a known) aminoacid sequence of the protein. A protein or a variant thereof can benaturally occurring or recombinant. Methods for detection and/ormeasurement of polypeptides in biological material are well known in theart and include, but are not limited to, Western-blotting, flowcytometry, ELISAs, RIAs, and various proteomics techniques. An exemplarymethod to measure or detect a polypeptide is an immunoassay, such as anELISA. This type of protein quantitation can be based on an antibodycapable of capturing a specific antigen, and a second antibody capableof detecting the captured antigen. Exemplary assays for detection and/ormeasurement of polypeptides are described in Harlow, E. and Lane, D.Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor LaboratoryPress.

The term “sequence identity,” as used herein, refers to the amount ofnucleotide which match exactly between two different sequences. Whencomparing RNA and DNA sequences Uracil and Thymine bases are consideredto be the same base. Gaps are not counted and the measurement istypically in relation to the shorter of the two sequences. For example:

A: AAGGCTT B: AAGGC C: AAGGCATHere identity (A,B)=100% (5 identicalnucleotides/min(length(A),length(B))). Identity(B,C)=100%, butidentity(A,C)=85% ((6 identical nucleotides/7). So 100% identity doesnot mean two sequences are the same.

The term “sequence similarity,” as used herein, can be described as anoptimal matching problem that finds the minimal number of editoperations (inserts, deletes, and substitutions) in order to transformthe one sequence into an exact copy of the other sequence being aligned(edit distance). Using this, the percentage sequence similarity of theexamples above are sim(A,B)=60%, sim(B,C)=60%, sim(A,C)=86%(semi-global, sim=1-(edit distance/unaligned length of the shortersequence)).

A “subject” in need thereof, refers to an individual who has a disease,a symptom of the disease, or a predisposition toward the disease, withthe purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disease, the symptom of the disease,or the predisposition toward the disease. In some embodiments, thesubject has hypercholesterolemia. In some embodiments, the subject hasatherosclerotic vascular disease. In some embodiments, the subject hashypertriglyceridemia. In some embodiments, the subject has diabetes. Theterm “subject” or “patient” encompasses mammals. Examples of mammalsinclude, but are not limited to, any member of the mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike.

The term “condition,” as used herein, includes diseases, disorders, andsusceptibilities. In some embodiments, the condition is anatherosclerotic vascular disease. In some embodiments, the condition isa hypertriglyceridemia. In some embodiments, the condition is adiabetes.

The term “atherosclerosis” or “atherosclerotic vascular disease,” asused herein, refers to a disease in which the inside of an arterynarrows due to the buildup of plaque. In some embodiments, it may resultin coronary artery disease, stroke, peripheral artery disease, or kidneyproblems.

The term “hypertriglyceridemia,” as used herein, refers to high (hyper-)blood levels (-emia) of triglycerides, the most abundant fatty moleculein most organisms. In some embodiments, elevated levels of triglyceridesare associated with atherosclerosis, even in the absence ofhypercholesterolemia (high cholesterol levels), and predispose tocardiovascular disease. In some embodiments, very high triglyceridelevels increase the risk of acute pancreatitis. In some embodiments,hypertriglyceridemia is associated with overeating, obesity, diabetesmellitus and insulin resistance, excess alcohol consumption, kidneyfailure, nephrotic syndrome, genetic predisposition (e.g., familialcombined hyperlipidemia, i.e., Type II hyperlipidemia), lipoproteinlipase deficiency, lysosomal acid lipase deficiency, cholesteryl esterstorage disease, certain medications (e.g., isotretinoin,hydrochlorothiazide diuretics, beta blockers, protease inhibitors),hypothyroidism (underactive thyroid), systemic lupus erythematosus andassociated autoimmune responses, glycogen storage disease type 1,propofol, or HIV medications.

The term “diabetes,” as used herein, refers to a group of metabolicdisorders characterized by a high blood sugar level over a prolongedperiod of time. In some embodiments, diabetes is type 1 diabetes thatresults from the pancreas's failure to produce enough insulin due toloss of beta cells. In some embodiments, diabetes is type 2 diabetescharacterized by insulin resistance, a condition in which cells fail torespond to insulin properly. In some embodiments, diabetes isgestational diabetes that occurs when pregnant women without a previoushistory of diabetes develop high blood sugar levels.

The term “low-density lipoprotein (LDL),” as used herein, refers to amicroscopic blob made up of an outer rim of lipoprotein and acholesterol center. In some embodiments, LDL has a highly hydrophobiccore composed of a polyunsaturated fatty acid known as linoleate andhundreds to thousands esterified and unesterified cholesterol molecules.In some embodiments, the core of LDL also carries triglycerides andother fats and is surrounded by a shell of phospholipids andunesterified cholesterol.

The term “high-density lipoprotein (HDL),” as used herein, refers to thesmallest lipoprotein particles. In embodiments, plasma enzymelecithin-cholesterol acyltransferase (LCAT) converts the freecholesterol into cholesteryl, which is then sequestered into the core ofthe lipoprotein particle, eventually causing the newly synthesized HDLto assume a spherical shape. In embodiments, HDL particles increase insize as they circulate through the bloodstream and incorporate morecholesterol and phospholipid molecules from cells and otherlipoproteins.

The term “cholesterol,” as used herein, refers to a lipid with a uniquestructure composed of four linked hydrocarbon rings forming the bulkysteroid structure. The term “triglyceride,” as used herein, refers to atri-ester composed of a glycerol bound to three fatty acid molecules. Insome embodiments, the fatty acids are saturated or unsaturated fattyacids.

The terms “treat,” “treating,” or “treatment,” and its grammaticalequivalents as used herein, can include alleviating, abating, orameliorating at least one symptom of a disease or a condition,preventing additional symptoms, inhibiting the disease or the condition,e.g., delaying, decreasing, suppressing, attenuating, diminishing,arresting, or stabilizing the development or progression of a disease orthe condition, relieving the disease or the condition, causingregression of the disease or the condition, relieving a condition causedby the disease or the condition, reducing disease severity, or stoppingthe symptoms of the disease or the condition either prophylacticallyand/or therapeutically. “Treating” also includes lessening the frequencyof occurrence or recurrence, or the severity, of any symptoms or otherill effects related to a disease or condition and/or the side effectsassociated with the disease or condition. “Treating” does notnecessarily require curative results. It is appreciated that, althoughnot precluded, treating a disorder or condition also does not requirethat the disorder, condition, or symptoms associated therewith becompletely eliminated. The term “treating” encompasses the concept of“managing” which refers to reducing the severity of a particular diseaseor disorder in a patient or delaying its recurrence, e.g., lengtheningthe period of remission in a patient who had suffered from the disease.“Treating” may refer to the application or administration or acomposition to a subject after the onset, or suspected onset, of adisease or condition.

The term “treating” further encompasses the concept of “prevent,”“preventing,” and “prevention.” The terms “prevent,” “preventing,” and“prevention,” as used herein, refer to a decrease in the occurrence ofpathology of a condition in a subject, who does not have, but is at riskof or susceptible to developing a disease or condition. The preventionmay be complete, e.g., the total absence of pathology of a condition ina subject. The prevention may also be partial, such that the occurrenceof pathology of a condition in a subject is less than that which wouldhave occurred without the present disclosure.

By “treating or preventing a condition,” for example, as compared withan equivalent untreated control, alleviating a symptom of a disorder mayinvolve reduction or degree of prevention at least 3%, 5%, 10%, 20%,40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% asmeasured by any standard technique. In some embodiments, alleviating asymptom of a disorder may involve reduction or degree of prevention byat least 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, or 10000 fold as compared with an equivalent untreatedcontrol.

As used therein, “delaying” the development of a disease means to defer,hinder, slow, retard, stabilize, and/or postpone progression of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individuals being treated. A method that“delays” or alleviates the development of a disease, or delays the onsetof the disease, is a method that reduces probability of developing oneor more symptoms of the disease in a given time frame and/or reducesextent of the symptoms in a given time frame, when compared to not usingthe method. Such comparisons are typically based on clinical studies,using a number of subjects sufficient to give a statisticallysignificant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset.

As used herein “onset” or “occurrence” of a disease includes initialonset and/or recurrence.

“Administering” and its grammatical equivalents as used herein can referto providing pharmaceutical compositions described herein to a subjector a patient. Conventional methods, known to those of ordinary skill inthe art of medicine, can be used to administer the composition to thesubject, depending upon the type of disease to be treated or the site ofthe disease. For example, the composition can be administered, e.g.,orally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, via an implanted reservoir, or via infusion. One ormore such routes can be employed.

The term “parenteral” as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intraperitoneal,intradermal, intraarterial, intrasynovial, intrastemal, intrathecal,intravascular, intralesional, and intracranial injection or infusiontechniques. In addition, it can be administered to the subject viainjectable depot routes of administration such as using 1-, 3-, or6-month depot injectable or biodegradable materials and methods.

By “co-administering” is meant administering one or more additionaltherapeutic regimens or agents or treatments and the composition of thedisclosure sufficiently close in time to enhance the effect of one ormore additional therapeutic agents, or vice versa. In this regard, thecomposition of the disclosure described herein can be administeredsimultaneously with one or more additional therapeutic regimens oragents or treatments, at a different time, or on an entirely differenttherapeutic schedule (e.g., the first treatment can be daily, while theadditional treatment is weekly). For example, in embodiments, thesecondary therapeutic regimens or agents or treatments are administeredsimultaneously, prior to, or subsequent to the composition of thedisclosure.

The terms “pharmaceutical composition” and its grammatical equivalentsas used herein can refer to a mixture or solution comprising atherapeutically effective amount of an active pharmaceutical ingredienttogether with one or more pharmaceutically acceptable excipients,carriers, and/or a therapeutic agent to be administered to a subject,e.g., a human in need thereof.

The term “pharmaceutically acceptable” and its grammatical equivalentsas used herein can refer to an attribute of a material which is usefulin preparing a pharmaceutical composition that is generally safe,non-toxic, and neither biologically nor otherwise undesirable and isacceptable for veterinary as well as human pharmaceutical use.“Pharmaceutically acceptable” can refer a material, such as a carrier ordiluent, which does not abrogate the biological activity or propertiesof the compound, and is relatively nontoxic, i.e., the material may beadministered to a subject without causing undesirable biological effectsor interacting in a deleterious manner with any of the components of thepharmaceutical composition in which it is contained.

A “pharmaceutically acceptable excipient, carrier, or diluent” refers toan excipient, carrier, or diluent that can be administered to a subject,together with an agent, and which does not destroy the pharmacologicalactivity thereof and is nontoxic when administered in doses sufficientto deliver a therapeutic amount of the agent.

A “pharmaceutically acceptable salt” may be an acid or base salt that isgenerally considered in the art to be suitable for use in contact withthe tissues of human beings or animals without excessive toxicity,irritation, allergic response, or other problem or complication. Suchsalts include mineral and organic acid salts of basic residues such asamines, as well as alkali or organic salts of acidic residues such ascarboxylic acids. Specific pharmaceutical salts include, but are notlimited to, salts of acids such as hydrochloric, phosphoric,hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic,formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethanedisulfonic, 2-hydroxyethyl sulfonic, nitric, benzoic, 2-acetoxybenzoic,citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic,pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic,phenylacetic, alkanoic such as acetic, HOOC—(CH₂)_(n)—COOH where n is0-4, and the like. Similarly, pharmaceutically acceptable cationsinclude, but are not limited to sodium, potassium, calcium, aluminum,lithium and ammonium. Those of ordinary skill in the art will recognizefrom this disclosure and the knowledge in the art that furtherpharmaceutically acceptable salts include those listed by Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,p. 1418 (1985). In general, a pharmaceutically acceptable acid or basesalt can be synthesized from a parent compound that contains a basic oracidic moiety by any conventional chemical method. Briefly, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin an appropriate solvent.

The term “therapeutic agent” can refer to any agent that, whenadministered to a subject, has a therapeutic, diagnostic, and/orprophylactic effect and/or elicits a desired biological and/orpharmacological effect. Therapeutic agents can also be referred to as“actives” or “active agents.” Such agents include, but are not limitedto, cytotoxins, radioactive ions, chemotherapeutic agents, smallmolecule drugs, proteins, and nucleic acids.

“A therapeutically effective amount” as used herein refers to the amountof each composition of the present disclosure required to confertherapeutic effect on the subject, either alone or in combination withone or more other therapeutic agents. Hence, as used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, composition, therapeutic agent,prophylactic agent, etc.) that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, improve symptoms of, diagnose, prevent, and/ordelay the onset of the disease, disorder, and/or condition. In terms oftreatment, a “therapeutically effective amount” is an amount that issufficient to palliate, ameliorate, stabilize, reverse or slow theprogression of a disease or a condition, e.g., an atheroscleroticvascular disease, hypertriglyceridemia, or diabetes. A “therapeuticallyeffective amount” varies, as recognized by those skilled in the art,depending on the particular condition being treated, the severity of thecondition, the individual subject parameters including age, physicalcondition, size, gender and weight, the duration of the treatment, thenature of concurrent therapy (if any), the specific route ofadministration and like factors within the knowledge and expertise ofthe health practitioner. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation. It is generally preferred that a maximum dose of theindividual components or combinations thereof be used, that is, thehighest safe dose according to sound medical judgment. It will beunderstood by those of ordinary skill in the art, however, that asubject may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.Additionally, other medication the patient may be receiving will affectthe determination of the therapeutically effective amount of thetherapeutic agent to administer. Empirical considerations, such as thehalf-life, generally will contribute to the determination of the dosage.A “therapeutically effective amount” may be of any of the compositionsof the disclosure used alone or in conjunction with one or more agentsused to treat a condition. A therapeutically effective amount can beadministered in one or more administrations.

An effective initial method to determine a “therapeutically effectiveamount” may be by carrying out cell culture assays (for example, usingneuronal cells) or using animal models (for example, mice, rats,rabbits, dogs or pigs). A dose may be formulated in animal models toachieve a concentration range that includes the IC50 (i.e., theconcentration of the composition which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Inaddition to determining the appropriate concentration range for andisclosure composition to be therapeutically effective, animal modelsmay also yield other relevant information such as preferable routes ofadministration that will give maximum effectiveness. Adjusting the doseto achieve maximal efficacy in humans based on the methods describedabove and other methods is well within the capabilities of theordinarily skilled artisan.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50,as well as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

The term “protospacer,” or “target sequence” and their grammaticalequivalents as used herein can refer to a DNA sequence of a target gene.In the native state, a protospacer is adjacent to a PAM (protospaceradjacent motif). The term “spacer” can be the RNA version of theprotospacer that binds to the complementary strand of the protospacer. Aspacer can be within a guide RNA (gRNA). The site of cleavage by anRNA-guided nuclease is within a protospacer sequence. Please see FIG. 1Afor an illustration.

The term “base editing,” “gene editing,” or “gene modification” and itsgrammatical equivalents as used herein can refer to genetic engineeringin which one or more nucleotides are inserted, replaced, or removed froma genome. Gene editing can be performed using a nuclease (e.g., anatural-existing nuclease or an artificially engineered nuclease). Genemodification can include introducing a double stranded break, anon-sense mutation, a frameshift mutation, a splice site alteration, oran inversion in a polynucleotide sequence, e.g., a target polynucleotidesequence.

The term “base editor (BE)” or “nucleobase editor (NBE)” as used hereincan refer to an agent that binds a polynucleotide and has nucleobasemodifying activity. In various embodiments, the base editor comprises anucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acidprogrammable nucleotide binding domain in conjunction with a guidepolynucleotide (e.g., guide RNA), or nucleic acids encoding theprogrammable nucleotide binding domain and the deaminase. In variousembodiments, the agent is a biomolecular complex comprising a proteindomain having base editing activity, i.e., a domain capable of modifyinga base (e.g., A, T, C, G, or U) within a nucleic acid molecule (e.g.,DNA), or a nucleic acid encoding the same. In some embodiments, thepolynucleotide programmable DNA binding domain is fused or linked to adeaminase domain, resulting in a base editor fusion protein. In someembodiments, the base editor comprises a nucleic acid encoding the baseeditor fusion protein, e.g., a mRNA encoding the base editor fusionprotein. The base editor fusion protein may comprise one or morelinkers, for example, peptide linkers. In one embodiment, the agent is afusion protein comprising a domain having base editing activity. Inanother embodiment, the protein domain having base editing activity islinked to the guide RNA (e.g., via an RNA binding motif on the guide RNAand an RNA binding domain fused to the deaminase). In some embodiments,the domain having base editing activity is capable of deaminating a basewithin a nucleic acid molecule. In some embodiments, the base editor iscapable of deaminating one or more bases within a DNA molecule. In someembodiments, the base editor is capable of deaminating an adenosine (A)within DNA. In some embodiments, the base editor is an adenosine baseeditor (ABE). In some embodiments, the base editor is capable ofdeaminating an cytosine (C) within DNA. In some embodiments, the baseeditor is a cytosine base editor (CBE).

The term “base editor system” refers to a system for editing anucleobase of a target nucleotide sequence. In various embodiments, thebase editor system comprises (1) a polynucleotide programmablenucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., anadenosine deaminase or a cytidine deaminase) for deaminating saidnucleobase; and (3) one or more guide polynucleotide (e.g., guide RNA).In some embodiments, the base editor system comprises a base editorfusion protein comprising (1) and (2). In some embodiments, thepolynucleotide programmable nucleotide binding domain is apolynucleotide programmable DNA binding domain. In some embodiments, thebase editor is an adenine or adenosine base editor (ABE). In someembodiments, the base editor is a cytosine base editor (CBE).

Nucleobase Editor Systems

In some aspects, provided herein are base editor systems capable ofnucleobase modifications. In some embodiments, the base editor systemcomprises (i) a guide polynucleotide or a nucleic acid encoding same,and (ii) a base editor fusion protein comprising a programmable DNAbinding domain and a deaminase, or a nucleic acid encoding same. In someembodiments, the base editor system comprises a guide polynucleotide. Insome embodiments, the base editor system comprises a nucleic acidencoding a guide polynucleotide. In some embodiments, the base editorsystem comprises a base editor fusion protein comprising a programmableDNA binding domain and a deaminase. In some embodiments, the base editorsystem comprises a nucleic acid encoding a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase.

In some embodiments, the guide polynucleotide directs the base editorsystem to effect a nucleobase alteration in a PCSK9 or ANGPTL3 gene invivo when administered to a subject.

In some embodiments, the base alteration occurs in at least 35% of wholeliver cells in the subject as measured by next generation sequencing orSanger sequencing.

In some embodiments, the base alteration occurs in at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% of whole liver cells in the subject asmeasured by next generation sequencing or Sanger sequencing. In someembodiments, the base alteration occurs in at most 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%,99.8%, or 99.9% of whole liver cells in the subject as measured by nextgeneration sequencing or Sanger sequencing. In some embodiments, thebase alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%,5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%,20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%,50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%,80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of whole liver cells in thesubject as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%,1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%,1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%,1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%,or 1%-2% of whole liver cells in the subject as measured by nextgeneration sequencing or Sanger sequencing. In some embodiments, thebase alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%,25%-65%, 30%-60%, 35%-55%, or 40%-50% of whole liver cells in thesubject as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 100% of whole livercells in the subject as measured by next generation sequencing or Sangersequencing.

In some embodiments, the base alteration occurs in hepatocytes in thesubject. In some embodiments, the base alteration occurs in at least 30%of hepatocytes in the subject as measured by next generation sequencingor Sanger sequencing. In some embodiments, the base alteration occurs inhepatocytes in the subject. In some embodiments, the base alterationoccurs in at least % of hepatocytes in the subject as measured by nextgeneration sequencing or Sanger sequencing. In some embodiments, thebase alteration occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9%of hepatocytes in the subject as measured by next generation sequencingor Sanger sequencing. In some embodiments, the base alteration occurs inat most 1%, 2%, 3%, 4%, 5%0, 10%, 15%, 20%, 25%, 30%, 3500 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% of hepatocytes in the subject as measuredby next generation sequencing or Sanger sequencing. In some embodiments,the base alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%,5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%,20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%,50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%,80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of hepatocytes in thesubject as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%,1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%,1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%,1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%,or 1%-2% hepatocytes in the subject as measured by next generationsequencing or Sanger sequencing. In some embodiments, the basealteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%,30%-60%, 35%-55%, or 40%-50% of hepatocytes in the subject as measuredby next generation sequencing or Sanger sequencing. In some embodiments,the base alteration occurs in 100% of hepatocytes in the subject asmeasured by next generation sequencing or Sanger sequencing.

In some embodiments, the base alteration occurred in whole liver cellsin the subject is measured by next generation sequencing. In someembodiments, the base alteration occurred in whole liver cells in thesubject is measured by Sanger sequencing. In some embodiments, the basealteration occurred in hepatocytes in the subject is measured by nextgeneration sequencing. In some embodiments, the base alteration occurredin hepatocytes in the subject is measured by Sanger sequencing.

In some embodiments, the nucleobase alteration results in a reduction ofat least 35% in blood PCSK9 protein level in the subject as compared toprior to the administration as measured by ELISA, Western blots, orLC-MS/MS. In some embodiments, the nucleobase alteration results in areduction of at least 35% in blood ANGPTL3 protein level in the subjectas compared to prior to the administration as measured by ELISA, Westernblots, or LC-MS/MS

In some embodiments, the nucleobase alteration results in a reduction ofat least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% in blood PCSK9 protein level in the subjectas compared to prior to the administration as measured by ELISA, Westernblots, or LC-MS/MS. In some embodiments, the nucleobase alterationresults in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% in blood PCSK9 protein level in thesubject as compared to prior to the administration as measured by ELISA,Western blots, or LC-MS/MS. In some embodiments, the nucleobasealteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%,99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood PCSK9 protein level in thesubject as compared to prior to the administration as measured by ELISA,Western blots, or LC-MS/MS. In some embodiments, the nucleobasealteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%,4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%,15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 31%-99.9%, 32%-99.9%,33%-99.9%, 34%-99.9%, 35%-99.9%, 36%-99.9%, 37%-99.9%, 38%-99.9%,39%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%,65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or95-99.9% in blood PCSK9 protein level in the subject as compared toprior to the administration as measured by ELISA, Western blots, orLC-MS/MS. In some embodiments, the nucleobase alteration results in areduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%,1%-85%, 1%-80%, 1%-79%, 1%-78%, 1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%,1%-72%, 1%-71%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%,1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%,1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1-9%, 1%-8%, 1%-7%, 1%-6%,1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood PCSK9 protein level in thesubject as compared to prior to the administration as measured by ELISA,Western blots, or LC-MS/MS. In some embodiments, the nucleobasealteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%,15%-97%, 20%-95%, 25%-90%, 30%-85%, 31%-80%, 32%-79%, 33%-78%, 34%-77%,35%-76%, 36%-76%, 37%-75%, 38%-74%, 39%-73%, 40%-72%, 45%-71%, 50%-70%,or 55%-65% in blood PCSK9 protein level in the subject as compared toprior to the administration as measured by ELISA, Western blots, orLC-MS/MS. In some embodiments, the nucleobase alteration results in areduction of 100% in blood PCSK9 protein level in the subject ascompared to prior to the administration as measured by ELISA, Westernblots, or LC-MS/MS.

In some embodiments, the nucleobase alteration results in at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%,290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood PCSK9protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS. In some embodiments,the nucleobase alteration results in at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood PCSK9protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS.

In some embodiments, the reduction of blood PCSK9 protein level or theblood PCSK9 protein level in the subject as compared to prior to theadministration is measured by ELISA (enzyme-linked immunosorbent assay).In some embodiments, the reduction of blood PCSK9 protein level or theblood PCSK9 protein level in the subject as compared to prior to theadministration is measured by Western blot analysis. In someembodiments, the reduction of blood PCSK9 protein level or the bloodPCSK9 protein level in the subject as compared to prior to theadministration is measured by LC-MS/MS (liquid chromatography-tandemmass spectrometry).

In some embodiments, the nucleobase alteration results in a reduction ofat least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% in blood ANGPTL3 protein level in the subjectas compared to prior to the administration as measured by ELISA, Westernblots, or LC-MS/MS. In some embodiments, the nucleobase alterationresults in a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% in blood ANGPTL3 protein level in thesubject as compared to prior to the administration as measured by ELISA,Western blots, or LC-MS/MS. In some embodiments, the nucleobasealteration results in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 85%, 90%, 95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%,99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood ANGPTL3 protein level inthe subject as compared to prior to the administration as measured byELISA, Western blots, or LC-MS/MS. In some embodiments, the nucleobasealteration results in a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%,4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%,15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 31%-99.9%, 32%-99.9%,33%-99.9%, 34%-99.9%, 35%-99.9%, 36%-99.9%, 37%-99.9%, 38%-99.9%,39%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%,65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or95-99.9% in blood ANGPTL3 protein level in the subject as compared toprior to the administration as measured by ELISA, Western blots, orLC-MS/MS. In some embodiments, the nucleobase alteration results in areduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%,1%-85%, 1%-80%, 1%-79%, 1%-78%, 1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%,1%-72%, 1%-7%, 1%0-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%,1%-39%, 1%-38%, 1%-37%, 1%-36%, 1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%,1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%,1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood ANGPTL3 protein level in thesubject as compared to prior to the administration as measured by ELISA,Western blots, or LC-MS/MS. In some embodiments, the nucleobasealteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%,15%-97%, 20%-95%, 25%-90%, 30%-85%, 31%-80%, 32%-79%, 33%-78%, 34%-77%,35%-76%, 36%-76%, 37%-75%, 38%-74%, 39%-73%, 40%-72%, 45%-71%, 50%-70%,or 55%-65% in blood ANGPTL3 protein level in the subject as compared toprior to the administration as measured by ELISA, Western blots, orLC-MS/MS. In some embodiments, the nucleobase alteration results in areduction of 100% in blood ANGPTL3 protein level in the subject ascompared to prior to the administration as measured by ELISA, Westernblots, or LC-MS/MS.

In some embodiments, the nucleobase alteration results in at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%,290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood ANGPTL3protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS. In some embodiments,the nucleobase alteration results in at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood ANGPTL3protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS.

In some embodiments, the reduction of blood ANGPTL3 protein level or theblood ANGPTL3 protein level in the subject as compared to prior to theadministration is measured by ELISA (enzyme-linked immunosorbent assay).In some embodiments, the reduction of blood ANGPTL3 protein level or theblood ANGPTL3 protein level in the subject as compared to prior to theadministration is measured by Western blot analysis. In someembodiments, the reduction of blood ANGPTL3 protein level or the bloodANGPTL3 protein level in the subject as compared to prior to theadministration is measured by LC-MS/MS (liquid chromatography-tandemmass spectrometry).

In some embodiments, the nucleobase alteration results in a reduction ofat least 35% in blood or low-density lipoprotein cholesterol (LDL-C)levels in the subject as compared to prior to the administration. Insome embodiments, the nucleobase alteration results in a reduction of atleast 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% in blood low-density lipoprotein cholesterol(LDL-C) level in the subject as compared to prior to the administration.In some embodiments, the nucleobase alteration results in a reduction ofat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in bloodlow-density lipoprotein cholesterol (LDL-C) level in the subject ascompared to prior to the administration. In some embodiments, thenucleobase alteration results in a reduction of at most 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%,99.7%, 99.8%, or 99.9% in blood low-density lipoprotein cholesterol(LDL-C) level in the subject as compared to prior to the administration.In some embodiments, the nucleobase alteration results in a reduction of1%-990.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%,8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%,30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%,60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%,90%-99.9%, or 95-99.9% in blood low-density lipoprotein cholesterol(LDL-C) level in the subject as compared to prior to the administration.In some embodiments, the nucleobase alteration results in a reduction of1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%,1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%,1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%,1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood low-density lipoproteincholesterol (LDL-C) level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%,25%-90%, 30%-85%, 35%-80%, 40%-75%, 45%-70%, 50%-65%, or 55%-60% inblood low-density lipoprotein cholesterol (LDL-C) level in the subjectas compared to prior to the administration. In some embodiments, thenucleobase alteration results in a reduction of 100% in bloodlow-density lipoprotein cholesterol (LDL-C) level in the subject ascompared to prior to the administration. In some embodiments, thenucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9% 10%, 11%, 2%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%,600%, 700%, 800%, 900%, 1000% less blood low-density lipoproteincholesterol (LDL-C) level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, ormore than 10-fold less blood low-density lipoprotein cholesterol (LDL-C)level in the subject as compared to prior to the administration.

In some embodiments, the nucleobase alteration results in a reduction ofat least 35% in blood triglyceride levels in the subject as compared toprior to the administration. In some embodiments, the nucleobasealteration results in a reduction of at least 30%, 35%, 40%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%in blood triglyceride level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%7, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or99.9% in blood triglyceride level in the subject as compared to prior tothe administration. In some embodiments, the nucleobase alterationresults in a reduction of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9%in blood triglyceride level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%,6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%,25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%,55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%,85%-99.9%, 90%-99.9%, or 95-99.9% in blood triglyceride level in thesubject as compared to prior to the administration. In some embodiments,the nucleobase alteration results in a reduction of 1%-99.5%, 1%-99%,1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%,1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%,1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%,or 1%-2% in blood triglyceride level in the subject as compared to priorto the administration. In some embodiments, the nucleobase alterationresults in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%,25%-90%, 30%-85%, 35%-80%, 40%-75%, 45%-70%, 50%-65%, or 55%-60% inblood triglyceride level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of 100% in blood triglyceride level in the subject ascompared to prior to the administration. In some embodiments, thenucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%,600%, 700%, 800%, 900%, 1000% less blood triglyceride level in thesubject as compared to prior to the administration. In some embodiments,the nucleobase alteration results in at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less bloodtriglyceride level in the subject as compared to prior to theadministration.

In some embodiments, the blood triglyceride level or the reduction ofblood triglyceride level in the subject as compared to prior to theadministration is measured by any standard technique. In someembodiments, the blood low-density lipoprotein cholesterol (LDL-C) levelor the reduction of blood low-density lipoprotein cholesterol (LDL-C)level in the subject as compared to prior to the administration ismeasured by any standard technique. For example, a clinical analyzerinstrument may be used to measure a ‘lipid panel’ in serum samples whichentails the direct measurement of cholesterol (total C), triglycerides(TG) and high-density lipoprotein cholesterol (HDL-C) enzymatically.Reagent kits specific for each analyte contain buffers, calibrators,blanks and controls. As used in the present disclosure, cholesterol,triglycerides and HDL-C may be quantified using absorbance measurementsof specific enzymatic reaction products. LDL-C may be determinedindirectly. In some instances, most of circulating cholesterol can befound in three major lipoprotein fractions: very low-densitylipoproteins (VLDL), LDL and HDL. In some embodiments, total circulatingcholesterol may be estimated with the formula [TotalC]=[VLDL-C]+[LDL-C]+[HDL-C]. Thus the LDL-C can be calculated frommeasured values of total cholesterol, triglycerides and HDL-C accordingto the relationship: [LDL-C]=[total C]-[HDL-C]-[TG]/5, where [TG]/5 isan estimate of VLDL-cholesterol. A reagent kit specific fortriglycerides containing buffers, calibrators, blanks and controls maybe used. As used herein, serum samples from the study may be analyzedand triglycerides may be measured using a series of coupled enzymaticreactions. In some embodiments, H₂O₂ may be used to quantify theanalyte.as the end product of the last one and its absorbance at 500 nm,and the color intensity is proportional to triglyceride concentrations.

In some embodiments, the guide polynucleotide is a guide RNA, whereinthe guide RNA comprises a spacer sequence that binds to thecomplementary strand of a protospacer sequence of the PCSK9 gene with 0,1, or 2 mismatches. In some embodiments, the guide RNA comprises aspacer sequence that binds to the complementary strand of a protospacersequence of the PCSK9 gene with no mismatches. In some embodiments, theguide RNA comprises a spacer sequence that binds to the complementarystrand of a protospacer sequence of the PCSK9 gene with 1 mismatch. Insome embodiments, the guide RNA comprises a spacer sequence that bindsto the complementary strand of a protospacer sequence of the PCSK9 genewith 2 mismatches. In some embodiments, the guide RNA comprises a spacersequence that binds to the complementary strand of a protospacersequence of the PCSK9 gene with 3 mismatches. In some embodiments, theguide RNA comprises a spacer sequence that binds to the complementarystrand of a protospacer sequence of the PCSK9 gene with 4 mismatches. Insome embodiments, the guide RNA comprises a spacer sequence that bindsto the complementary strand of a protospacer sequence of the PCSK9 genewith 5 mismatches.

In some embodiments, the guide polynucleotide is a guide RNA, whereinthe guide RNA comprises a spacer sequence that binds to thecomplementary strand of a protospacer sequence of the ANGPTL3 gene with0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises aspacer sequence that binds to the complementary strand of a protospacersequence of the ANGPTL3 gene with no mismatches. In some embodiments,the guide RNA comprises a spacer sequence that binds to thecomplementary strand of a protospacer sequence of the ANGPTL3 gene with1 mismatch. In some embodiments, the guide RNA comprises a spacersequence that binds to the complementary strand of a protospacersequence of the ANGPTL3 gene with 2 mismatches. In some embodiments, theguide RNA comprises a spacer sequence that binds to the complementarystrand of a protospacer sequence of the ANGPTL3 gene with 3 mismatches.In some embodiments, the guide RNA comprises a spacer sequence thatbinds to the complementary strand of a protospacer sequence of theANGPTL3 gene with 4 mismatches. In some embodiments, the guide RNAcomprises a spacer sequence that binds to the complementary strand of aprotospacer sequence of the ANGPTL3 gene with 5 mismatches.

In some embodiments, the nucleobase alteration is outside of theprotospacer sequence in less than 1% of whole liver cells in the subjectas measured by net nucleobase editing. In some embodiments, thenucleobase alteration is outside of the protospacer sequence in lessthan 1% of hepatocytes in the subject as measured by net nucleobaseediting. In some embodiments, the nucleobase alteration is only withinthe protospacer sequence as measured by net nucleobase editing.

In some embodiments, the nucleobase alteration is outside of theprotospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%,85%, 90% of whole liver cells in the subject as measured by netnucleobase editing. In some embodiments, the nucleobase alteration isoutside of the protospacer sequence in less than 0.01%. 0.02%, 0.03%0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%,9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,65%, 80%, 85%, 90% of hepatocytes in the subject as measured by netnucleobase editing. In some embodiments, the nucleobase alteration isoutside of the protospacer sequence in less than 0.01%. 0.02%, 0.03%0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%,9.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,65%, 80%, 85%, 90% of cells in the subject as measured by net nucleobaseediting.

In some embodiments, the deaminase is an adenine deaminase. In someembodiments, the nucleobase alteration is a A•T to G•C alteration. Insome embodiments, the deaminase is an adenine deaminase and thenucleobase alteration is a A•T to G•C alteration. In some embodiments,the programmable DNA binding domain comprises a nuclease inactive Cas9or a Cas9 nickase. In some embodiments, the programmable DNA bindingdomain comprises a Cas9.

In some embodiments, the nucleobase alteration is at a splice site ofthe PCSK9 gene. In some embodiments, the nucleobase alteration is at asplice donor site of the PCSK9 gene. In some embodiments, the splicedonor site is at 5′ end of PCSK9 intron 1 as referenced in SEQ ID NO: 5.In some embodiments, the nucleobase alteration is at a splice acceptorsite of the PCSK9 gene. In some embodiments, the nucleobase alterationresults in a frame shift, a premature stop codon, a insertion ordeletion in a transcript encoded by the PCSK9 gene. In some embodiments,the nucleobase alteration results in an aberrant transcript encoded bythe PCSK9 gene. In some embodiments, the guide polynucleotide is a guideRNA. In some embodiments, the guide RNA is chemically modified. In someembodiments, the guide RNA comprises a tracrRNA sequence. In someembodiments, the guide RNA comprises a chemical modification as setforth in Table 1 or Table 24.

In some embodiments, the nucleobase alteration is at a splice site ofthe ANGPTL3 gene. In some embodiments, the nucleobase alteration is at asplice donor site of the ANGPTL3 gene. In some embodiments, the splicedonor site is at 5′ end of ANGPTL3 intron 6 as referenced in SEQ ID NO:7. In some embodiments, the nucleobase alteration is at a spliceacceptor site of the ANGPTL3 gene. In some embodiments, the nucleobasealteration results in a frame shift, a premature stop codon, a insertionor deletion in a transcript encoded by the ANGPTL3 gene. In someembodiments, the nucleobase alteration results in an aberrant transcriptencoded by the ANGPTL3 gene. In some embodiments, the guidepolynucleotide is a guide RNA. In some embodiments, the guide RNA ischemically modified. In some embodiments, the guide RNA comprises atracrRNA sequence. In some embodiments, the guide RNA comprises achemical modification as set forth in Table 1 or Table 24.

In some embodiments, the guide RNA comprises a guide RNA sequence setforth in Table 1 or Table 24. In some embodiments, the guide RNAcomprises the sequence5′-5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUC cGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3′ (SEQ ID NO: 9),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9) (GA346),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAacAAcuugaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 10) (GA374),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuuu-3′ (SEQ ID NO: 11)(GA385),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuUu-3′ (SEQ ID NO: 11) (GA386)or 5′-cscscsGCACCUUGGCGCAGCGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcuususuuuu-3′ (SEQ ID NO: 12) (GA387).

In some embodiments, the protospacer sequence comprises a protospacersequence set forth in Table 1 or Table 24. In some embodiments, theprotospacer comprises the sequence

(SEQ ID NO: 13) 5′-CCCGCACCTTGGCGCAGCGG-3′, (SEQ ID NO: 14)AAGATACCTGAATAACTCTC-3′, and (SEQ ID NO: 15) 5′-AAGATACCTGAATAACCCTC-3′.

In some embodiments, the base editor fusion protein comprises thesequence of SEQ ID NO: 3. In some embodiments, the adenosine deaminasecomprises an amino acid sequence that is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO:3 or to any of the adenosine deaminases provided herein. It should beappreciated that adenosine deaminases provided herein may include one ormore mutations (e.g., any of the mutations provided herein). Thedisclosure provides any deaminase domains with a certain percentidentity plus any of the mutations or combinations thereof describedherein. In some embodiments, the adenosine deaminase comprises an aminoacid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,or more mutations compared to the amino acid sequence set forth in SEQID NO: 3 or any of the adenosine deaminases provided herein. In someembodiments, the adenosine deaminase comprises an amino acid sequencethat has at least 5, at least 10, at least 15, at least 20, at least 25,at least 30, at least 35, at least 40, at least 45, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least110, at least 120, at least 130, at least 140, at least 150, at least160, or at least 170 identical contiguous amino acid residues ascompared to any one of the amino acid sequences set forth in SEQ ID NO:3 or any of the adenosine deaminases provided herein.

In some embodiments, the nucleic acid encoding the base editor fusionprotein is a mRNA. The mRNA may comprise modifications, for example,modifications at 3′ or 5′ end of the mRNA. In some embodiments, the mRNAcomprises a cap analog.

In some embodiments, the mRNA comprises at least 1, 2, or 3 nucleotidesat the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, oradditional 2′ chemical modification or a combination thereof. In someembodiments, the mRNA comprises at least 1, 2, or 3 nucleotides at the5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, oradditional 2′ chemical modification or a combination thereof. In someembodiments, the mRNA comprises at least 1 nucleotide at the 5′ end thatcomprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′chemical modification or a combination thereof. In some embodiments, themRNA comprises at least 2 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 3 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 4 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 5 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 6 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 7 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 8 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 9 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 10 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof.

In some embodiments, the mRNA comprises a poly A tail. The poly A tailmay be at the 3′ end of the mRNA.

In some embodiments, the GC % content of the mRNA sequence is greaterthan or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In someembodiments, the GC % content of the mRNA sequence is greater than orequal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

In some embodiments, the mRNA sequence comprises an adenine tTNAdeaminase (TadA) region. In some embodiments, the GC % of the TadAregion is greater than or equal to 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89% or 90%. In some embodiments, the GC % content of the TadA region isgreater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

In some embodiments, the mRNA sequence comprises a Cas9 region. In someembodiments, the GC % of the Cas9 region is greater than or equal to40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, theGC % content of the Cas9 region is greater than or equal to 40%, 45%,50%, 55%, 60%, 65%, 70%, 75% or 80%.

In some embodiments, the mRNA sequence comprises a NLS region. In someembodiments, the GC % of the NLS region is greater than or equal to 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC %content of the NLS region is greater than or equal to 40%, 45%, 50%,55%, 60%, 65%, 70%, 75% or 80%.

In some embodiments, the mRNA sequence comprises a first linker regionthat connects the TadA region and the Cas9 region. In some embodiments,the GC % of the first linker region is greater than or equal to 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC %content of the first linker region is greater than or equal to 40%, 45%,50%, 55%, 60%, 65%, 70%, 75% or 80%.

In some embodiments, the mRNA sequence comprises a second linker regionthat connects the Cas9 region and the NLS region. In some embodiments,the GC % of the second linker region is greater than or equal to 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the GC %content of the second linker region is greater than or equal to 40%,45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%.

In some embodiments, the base editor system as provided herein furthercomprises a lipid nanoparticle (LNP) enclosing a guide polynucleotide ora nucleic acid encoding the guide polynucleotide (i). In someembodiments, the LNP further encloses a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same (ii). In some embodiments, the base editorsystem further comprises a second LNP enclosing a base editor fusionprotein comprising a programmable DNA binding domain and a deaminase, ora nucleic acid encoding same (ii).

A base editor system as provided herein can include one or more LNPs.For example, a base editor system may comprise a LNP enclosing both aguide polynucleotide and a nucleic acid encoding the base editor fusionprotein, e.g. an mRNA encoding the base editor fusion protein. Inanother example, a base editor system may comprise a LNP enclosing aguide polynucleotide, e.g. a guide RNA, and a LNP enclosing a nucleicacid, e.g. an mRNA, encoding the base editor fusion protein. LNPsseparately enclosing the guide polynucleotide and the base editor fusionprotein or mRNA encoding the base editor fusion protein may allow forflexible dosing and administration of the base editor system. Forexample, a LNP enclosing a guide RNA can be administered first, followedby administration of a LNP enclosing a mRNA encoding the base editorfusion protein. In some embodiments, a LNP enclosing a guide RNA and asecond LNP enclosing a mRNA encoding the base editor fusion protein areadministered to a subject at the same time. In some embodiments, a LNPenclosing a guide RNA and a LNP enclosing a mRNA encoding the baseeditor protein are administered to a subject sequentially. In someembodiments, a LNP enclosing mRNA encoding the base editor fusionprotein is administered to a subject, followed by multipleadministration or doses of a second LNP enclosing a guide RNA after 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,or 12 weeks or more. The multiple doses of the second LNP may beadministered with intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 days or more.

In some embodiments, the ratio of the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 1:10 toabout 10:1 by weight. In some embodiments, the ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 1:1, 1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, 1:4 or anyratio between 4:1 or 1:4 by weight. In some embodiments, the ratio ofthe guide polynucleotide and the nucleic acid encoding the base editorfusion protein can be determined by titration of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein.

In some embodiments, the molar ratio of the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 500:1 toabout 1:500.

In some embodiments, the ratio of the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 1000:1 toabout 1:1000 by weight. In some embodiments, the ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1,650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1,100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1,40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1,12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2; 1, 1.9:1, 1.8:1,1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1,0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In someembodiments, the ratio of the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3,1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19,1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75,1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400,1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900,1:950, or 1:1000 by weight. In some embodiments, the ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1,700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1,200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1,50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1,14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2; 1,1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1,0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 byweight. In some embodiments, the ratio of the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is at leastabout 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9,1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50,1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150,1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650,1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In someembodiments, the ratio of the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is at most about 1000:1, 950:1,900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1,400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1,70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1,18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2; 1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5,1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25,1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85,1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450,1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or1:1000 by weight.

In some embodiments, the molar ratio of the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 10000:1 toabout 1:10000. In some embodiments, the molar ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1,7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1,2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1,700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1,200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1,100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1,64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1,52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1,40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1,28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1,16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1,or 0.1:1. In some embodiments, the molar ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000,1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500,1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800,1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300,1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120,1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67,1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55,1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43,1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31,1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19,1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7,1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4,1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at least about 10000:1, 9500:1, 9000:1, 8500:1,8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1,3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1,800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1,300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1,120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1,67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1,55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1,43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1,31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1,19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1,0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio ofthe guide polynucleotide and the nucleic acid encoding the base editorfusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500,1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000,1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850,1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350,1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130,1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68,1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56,1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44,1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32,1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20,1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5,1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio ofthe guide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1,7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1,3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1,750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1,250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1,110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1,66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1,54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1,42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1,30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000,1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500,1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800,1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300,1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120,1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67,1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55,1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43,1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31,1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19,1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7,1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4,1:0.3, 1:0.2, or 1:0.1.

In some embodiments, the ratio of a nucleic acid encoding the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 10:1 to about 1:10 by weight. In some embodiments, theratio of a nucleic acid encoding the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 4:1, 3;1,2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is about 500:1to about 1:500.

In some embodiments, the ratio of a nucleic acid encoding the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 1000:1 to about 1:1000 by weight. In some embodiments,the ratio of a nucleic acid encoding the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 1000:1,950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1,450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1,75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1,19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of anucleic acid encoding the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3,1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19,1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75,1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400,1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900,1:950, or 1:1000 by weight. In some embodiments, the ratio of a nucleicacid encoding the guide polynucleotide and the nucleic acid encoding thebase editor fusion protein is at least about 1000:1, 950:1, 900:1,850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1,350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1,65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1,17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1,1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or0.1 by weight. In some embodiments, the ratio of a nucleic acid encodingthe guide polynucleotide and the nucleic acid encoding the base editorfusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5,1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25,1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85,1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450,1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or1:1000 by weight. In some embodiments, the ratio of a nucleic acidencoding the guide polynucleotide and the nucleic acid encoding the baseeditor fusion protein is at most about 1000:1, 950:1, 900:1, 850:1,800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1,300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1,60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1,17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1,1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 byweight. In some embodiments, the ratio of a nucleic acid encoding theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5,1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25,1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85,1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450,1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or1:1000 by weight.

In some embodiments, the molar ratio of a nucleic acid encoding theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is about 10000:1 to about 1:10000. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is about10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1,5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1,1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1,500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1,160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1,75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1,59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1,47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1,35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1,23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1,11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1,0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is at leastabout 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500,1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000,1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600,1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180,1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90,1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62,1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50,1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38,1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26,1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14,1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1,1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. Insome embodiments, the molar ratio of a nucleic acid encoding the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1,7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1,3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1,750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1,250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1,110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1,66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1,54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1,42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1,30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of anucleic acid encoding the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is at least about 1:10000,1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500,1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000,1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500,1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160,1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75,1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59,1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47,1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35,1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23,1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11,1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7,1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, themolar ratio of a nucleic acid encoding the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is at most about10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1,5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1,1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1,500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1,160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1,75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1,59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1,47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1,35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1,23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1,11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1,0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is at mostabout 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500,1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000,1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600,1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180,1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90,1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62,1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50,1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38,1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26,1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14,1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1,1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.

Precision genome editing is a growing field with industrial,agricultural, and biomedical applications. One of the dominantgenome-editing systems available today is clustered regularlyinterspaced short palindromic repeats (CRISPR)-CRISPR associated 9(Cas9). Through the use of a guide RNA (gRNA) with a sequence homologousto that of a sequence of DNA in the target genome (known as theprotospacer) adjacent to a specific protospacer-adjacent motif (PAM)comprising the sequence NGG (N is any standard base) in the DNA, Cas9can be used to create a double-strand break (DSB) at the targetedsequence. Non-homologous end joining (NHEJ) at DSBs can be used tocreate indels and knock out genes at genetic loci; likewise,homology-directed repair (HDR) can be used, with an introduced templateDNA, to insert genes or modify the targeted sequence. A variety ofCas9-based tools have been developed in recent years, including toolsthat methylate DNA, recognize broader sequence space, or createsingle-strand nicks. In 2016, Komor et al. described the use ofCRISPR-Cas9 to convert a cytosine base to a thymine base without theintroduction of a template DNA strand and without the need for DSBs(Komor A C, Kim Y B, Packer MS, et al. Programmable editing of a targetbase in genomic DNA without double-stranded DNA cleavage. Nature, 2016,533: 420-4, incorporated herein by reference in its entirety). After thecytidine deaminase domain of rat APOBEC1 was fused to the N-terminus ofcatalytically-dead Cas9 (dCas9) using the linker XTEN (resulting in afusion protein called base editor 1, or BE1), conversion of cytosine touracil was observed between position 4 and position 8 within the 20-ntprotospacer region of DNA (or, to express it a different way, 13 to 17nucleotides upstream of the PAM). Of note, any cytosine base within this“window” was amenable to editing, resulting in varied outcomes dependingon how many and which cytosines were edited. After DNA replication orrepair, each uracil was replaced by a thymine, completing the C to Tbase editing.3 The next version of base editor (BE2) incorporated auracil glycosylase inhibitor fused to the C-terminus of dCas9 to helpinhibit base excision repair of the uracil bases resulting from thecytidine deaminase activity (which otherwise would act to restore theoriginal cytosine bases); this improved the efficiency of C to T baseediting. The final version, BE3, used a Cas9 nickase rather than dCas9;the nickase cut the unedited strand opposite the edited C to T bases,stimulating the removal of the opposing guanidine through eukaryoticmismatch repair. BE2 and BE3 base editing was observed in both human andmurine cell lines. The specificity of base editing has been furtherimproved through the addition of mutations to the Cas9 nickase; insimilar fashion, Cas9 has been mutated to narrow the width of theediting window from approximately 5 nucleotides to as little as 1-2nucleotides (Rees H A, Komor A C, Yeh W H, et al. Improving the DNAspecificity and applicability of base editing through proteinengineering and protein delivery. Nat Commun, 2017, 8: 15790, Kim Y B,Komor A C, Levy J M, et al. Increasing the genome-targeting scope andprecision of base editing with engineered Cas9-cytidine deaminasefusions. Nat Biotechnol, 2017, 35: 371-6, each of which is incorporatedherein by reference in its entirety).

An alternative cytosine base editing platform is by linking theactivation-induced cytosine deaminase domain PmCDA1 to dCas9(Target-AID), they were able to demonstrate targeted C to T base editingin yeast. Furthermore, an alternative C to T editing strategy was alsodemonstrated without fusing a deaminase domain to Cas9; instead, a SH3(Src 3 homology) domain was added to the C-terminus of dCas9 while a SHL(SH3 interaction ligand) was added to PmCDA1.6 Optimization ofefficiency was achieved through the use of a Cas9 nickase rather thandCas9. Further, an uracil DNA glycosylase inhibitor was added to enhancebase editing in the mammalian CHO cell line. The resulting platform wasable to consistently edit bases within 3 to 5 bases of the 18thnucleotide upstream of the PAM sequence (Nishida K, Arazoe T, Yachie N,et al. Targeted nucleotide editing using hybrid prokaryotic andvertebrate adaptive immune systems. Science, 2016, 353: aaf8729,incorporated herein by reference in its entirety).

A distinct cytosine base editing platform used Cpf1 (also known asCas12a) instead of Cas9 as the RNA-guided endonuclease.Catalytically-inactive Cpf1 was fused to APOBEC1 (dLbCpfl-BE0), leadingto C to T conversion in a human cell line. While the Cas9 base editorvariants BE3 and Target-AID recognize the PAM sequence NGG, dLbCpfl-BE0recognizes the T-rich PAM sequence TTTV. Although base editing wasobserved between positions 8 and 13 of the protospacer sequence withdLbCpfl-BE0, the introduction of additional mutations into Cpf1 was ableto reduce the window to positions 10 to 12. However, narrowing of thebase editing window correlated with a decrease in editing efficiency (LiX, Wang Y, Liu Y, et al. Base editing with a Cpf1-cytidine deaminasefusion. Nat Biotechnol, 2018, 36: 324-7, incorporated herein byreference in its entirety).

Cytosine base editing is not wholly predictable; indels can occur at thetarget site, albeit at lower frequencies that those observed for C to Tediting editors. Furthermore, cytosine base editors can occasionallycause C to A or C to G edits rather than the expected C to T edits.Adding linker lengths between Cas9 nickase and the rat APOBEC1 cytosinedeaminase domain from 16 amino acids to 32 amino acids, the linkerbetween the Cas9 nickase and the uracil glycosylase inhibitor from 4amino acids to 9 amino acids, and using a second uracil glycosylaseinhibitor was appended to the C-terminus of the new cytosine base editorusing another 9 amino acid linker improved cytosine base editor termed“BE4” (Komor A C, Zhao K T, Packer M S, et al. Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:Abase editors with higher efficiency and product purity. Sci Adv, 2017,3: eaao4774. Incorporated herein by reference in its entirety).

By fusing Escherichia coli adenine tTNA deaminase TadA (ecTadA) to dCas9and mutagenesis of the ecTadA domain in conjunction with selection forediting activity revealed that A106V and D108N mutations yielded a baseeditor capable of editing adenine to guanine in DNA, termed ABE7.10(Gaudelli N M, Komor A C, Rees H A, et al. Programmable base editing ofA•T to G•C in genomic DNA without DNA cleavage. Nature, 2017, 551:464-71. Incorporated herein by reference in its entirety). Koblan et al.improved the efficiency of ABE7.10 through modification of nuclearlocalization signals and codon optimization, yielding a version calledABEmax; a similar approach improved the efficiency of the cytosine baseeditor BE4.10 Huang et al. performed further development of both adenineand cytosine base editors to use alternative PAMs and to expand theirediting windows, thereby increasing their targeting range (Improvingcytidine and adenine base editors by expression optimization andancestral reconstruction. Nat Biotechnol, 2018, 36: 843-6, Incorporatedherein by reference in its entirety).

The same has proven to be true of base editors, 12-14 althoughcomparisons of Cas9, cytosine base editors, and adenine base editorsusing the same gRNAs have shown distinct off-target profiles.

A variety of studies have raised concern about gRNA-independentoff-target base editing, caused by the deaminase domain acting inisolation (without the need for engagement of DNA by the Cas9-gRNAcomplex). Additional studies showed that the gRNA-independent off-targeteffects of base editors are not limited to DNA. RNA sequencing of cellstreated with either cytosine base editors or adenine base editorsrevealed transcriptome-wide off-target editing of RNA, and thatintroduction of amino acid substitution in the deaminase domain ofadenine base (e.g. R106W) editors reduced off-target editing of RNAwithout substantially reducing on-target DNA base-editing efficiency.Zuo E, Sun Y, Wei W, et al. Cytosine base editor generates substantialoff-target single-nucleotide variants in mouse embryos. Science, 2019,364: 289-92; Jin S, Zong Y, Gao Q, et al. Cytosine, but not adenine,base editors induce genome-wide off-target mutations in rice. Science,2019, 364: 292-5; Grunewald J, Zhou R, Garcia SP, et al.Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNAbase editors. Nature, 2019, 569: 433-7; Grünewald J, Zhou R, Iyer S, etal. CRISPR DNA base editors with reduced RNA off-target and self-editingactivities. Nat Biotechnol, 2019, 37: 1041-8; Zhou C, Sun Y, Yan R, etal. Off-target RNA mutation induced by DNA base editing and itselimination by mutagenesis. Nature, 2019, 571: 275-8; Rees H A, WilsonC, Doman J L, et al. Analysis and minimization of cellular RNA editingby DNA adenine base editors. Sci Adv, 2019, 5: eaax5717, each of whichis incorporated herein by reference in its entirety).

Correction of disease-causing mutations via precision editing withstandard Cas9 genome editing has largely required HDR. Since HDR islimited to cells in S or G2 phase of mitosis, precision editing ofnon-mitotic cells is difficult. However, base editors are not reliant onHDR; the editing of postmitotic cochlear cells in mice is feasible withcytosine base editor BE3. By injecting BE3 and a gRNA in the form of apreassembled ribonucleoprotein via cationic liposomes, serine-33 inbeta-catenin was edited to phenylalanine (TCT codon edited to TTT),allowing for the transdifferentiation of supporting cells into haircells. Base editing of cochlear tissue was confirmed via sequencing,showing an editing rate between 0.7% and 3.0% depending on the region ofthe cochlea. In contrast, standard Cas9 editing via HDR showednegligible signs of efficacy in cochlear cells. A variant of SaBE3,delivered into the liver via adeno-associated viral (AAV) vectors, wasreported to directly correct a pathogenic T to C mutation in the Pahgene with an editing rate as high as 29% and thereby treat the diseasephenylketonuria in adult mice. Adenine base editing has also beendemonstrated to generate mutations in mice. Yeh W H, Chiang H, Rees H A,et al. In vivo base editing of post-mitotic sensory cells. Nat Commun,2018, 9: 2184; Villiger L, Grisch-Chan H M, Lindsay H, et al. Treatmentof a metabolic liver disease by in vivo genome base editing in adultmice. Nat Med, 2018, 24: 1519-25; Ryu S M, Koo T, Kim K, et al. Adeninebase editing in mouse embryos and an adult mouse model of Duchennemuscular dystrophy. Nat Biotechnol, 2018, 36: 536-9; Song C Q, Jiang T,Richter M, et al. Adenine base editing in an adult mouse model oftyrosinaemia. Nat Biomed Eng, 2020, 4: 125-30; Pisciotta L, Favari E,Magnolo L, et al. Characterization of three kindreds with familialcombined hypolipidemia caused by loss-of-function mutations of ANGPTL3.Circ Cardiovasc Genet, 2012, 5: 42-50. each of which is incorporatedherein by reference in its entirety.

Provided herein are compositions of nucleobase editor systems thatcomprises nucleobase editor proteins, complexes, or compounds that iscapable of making a modification or conversion to a nucleobase (e.g., A,T, C, G, or U) within a target nucleotide sequence.

A nucleobase editor or a base editor (BE) refers to an agent comprisinga polypeptide that is capable of making a modification to a base (e.g.,A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). Insome embodiments, the base editor is capable of deaminating a basewithin a nucleic acid. In some embodiments, the base editor is capableof deaminating a base within a DNA molecule. In some embodiments, thebase editor is capable of deaminating an adenine (A) in DNA.

In some embodiments, the base editor comprises a fusion proteincomprising a programmable DNA binding protein fused to an adenosinedeaminase. In some embodiments, the base editor comprises a fusionprotein comprising a Cas9 protein and an adenosine deaminase. In someembodiments, the base editor is a Cas9 nickase (nCas9) fused to anadenosine deaminase. In some embodiments, the base editor is anuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In someembodiments, the base editor comprises a fusion protein comprising aprogrammable DNA binding protein fused to an cytidine deaminase. In someembodiments, the base editor comprises a fusion protein comprising aCas9 protein and an cytidine deaminase. In some embodiments, the baseeditor is a Cas9 nickase (nCas9) fused to an cytidine deaminase. In someembodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fusedto an cytidine deaminase. In some embodiments, the base editor furthercomprises, an inhibitor of base excision repair, for example, a UGIdomain. In some embodiments, the fusion protein comprises a Cas9 nickasefused to a deaminase and an inhibitor of base excision repair, such as aUGI or dISN domain. In some embodiments, the dCas9 domain of the fusionprotein comprises a D10A and a H840A mutation as numbered in the wildtype SpCas9 amino acid sequence. In some embodiments, the UGI comprisesthe following amino acid sequence:

>sp|P14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor (SEQ ID NO: 16)MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTS D APE YKPW ALVIQDS NGENKIKML

In some embodiments, a base editor system provided herein comprises abase editor fusion protein. For example, a base editor fusion proteinmay comprise a programmable DNA binding protein and a deaminase, e.g. anadenosine deaminase. In some embodiments, any of the fusion proteinsprovided herein are base editors. In some embodiments, the programmableDNA binding protein is a Cas9 domain, a Cpf1 domain, a CasX domain, aCasY domain, a Cas12b domain, a C2c2 domain, aC2c3 domain, or anArgonaute domain. In some embodiments, the programmable DNA bindingprotein is a Cas9 domain. The Cas9 domain may be any of the Cas9 domainsor Cas9 proteins (e.g., nuclease inactive Cas9 or Cas9 nickase, or aCas9 variant from any species) provided herein. In some embodiments, anyof the Cas9 domains or Cas9 proteins provided herein may be fused withany of the deaminases provided herein. In some embodiments, the baseeditor comprises a deaminase, e.g., an adenosine deaminase and aprogrammable DNA binding protein, e.g., a Cas9 domain joined via alinker. In some embodiments, the base editor comprises a fusion proteincomprising a deaminase, e.g., an adenosine deaminase and a programmableDNA binding protein, e.g., a Cas9 domain joined via a linker. In someembodiments, the linker is a peptide linker. In some embodiments, alinker is present between the deaminase domain and the Cas9 domain. Insome embodiments, an deaminase and a programmable DNA binding domain arefused via any of the peptide linkers provided herein. For example, anadenosine deaminase and a Cas9 domain may be fused via a linker thatcomprises between 1 and 200 amino acids. In some embodiments, theadenosine deaminase and the programmable DNA binding protein are fusedvia a linker that comprises from 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200,10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 80, 10 to 100,10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80,20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80,30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to100, 40 to 150, 40 to 200, 50 to 60 50 to 80, 50 to 100, 50 to 150, 50to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150,80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length.In some embodiments, the adenosine deaminase and the programmable DNAbinding protein are fused via a linker that comprises 4, 16, 32, or 104amino acids in length. In some embodiments, the adenosine deaminase andthe programmable DNA binding protein are fused via a linker thatcomprises the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 17),SGGS (SEQ ID NO: 18), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 19),SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 20), orGGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 21). In someembodiments, the adenosine deaminase and the programmable DNA bindingprotein are fused via a linker comprising the amino acid sequenceSGSETPGTSESATPES (SEQ ID NO: 17), which may also be referred to as theXTEN linker. In some embodiments, the linker is 24 amino acids inlength. In some embodiments, the linker comprises the amino acidsequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 23). In some embodiments,the linker is 40 amino acids in length. In some embodiments, the linkercomprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 24).

In some embodiments, the linker is 64 amino acids in length. In someembodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQID NO: 25). In some embodiments, the linker is 92 amino acids in length.In some embodiments, the linker comprises the amino acid sequencePGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 26).

In some embodiments, a base editor system provided herein comprises abase editor comprising a fusion protein comprising an inhibitor of baserepair. In some embodiments, a base editor comprises a fusion proteincomprising a cytidine deaminase and a programmable DNA binding domain,e.g. a Cas9 domain. In some embodiments, a base editor comprises afusion protein comprising an adenosine deaminase and a programmable DNAbinding domain, e.g. a Cas9 domain. In some embodiments, the base editoror the fusion protein further comprises an inhibitor of base repair(IBR). In some embodiments, the IBR comprises an inhibitor of inosinebase repair. In some embodiments, the IBR is an inhibitor of inosinebase excision repair. In some embodiments, the inhibitor of inosine baseexcision repair is a catalytically inactive inosine specific nuclease(dISN). In some embodiments, a dISN may inhibit (e.g., by sterichindrance) inosine removing enzymes from excising the inosine residuefrom DNA. For example, catalytically dead inosine glycosylases (e.g.,alkyl adenine glycosylase [AAG]) will bind inosine but will not createan abasic site or remove the inosine, thereby sterically blocking thenewly-formed inosine moiety from potential DNA damage/repair mechanisms.Thus, this disclosure contemplates a fusion protein comprising aprogrammable DNA binding protein and an adenosine deaminase furtherfused to a dISN. This disclosure contemplates a fusion proteincomprising any Cas9 domain, for example, a Cas9 nickase (nCas9) domain,a catalytically inactive Cas9 (dCas9) domain, a high fidelity Cas9domain, or a Cas9 domain with reduced PAM exclusivity. It should beunderstood that the use of a dISN may increase the editing efficiency ofa adenosine deaminase that is capable of catalyzing a A to I change. Forexample, fusion proteins comprising a dISN domain may be more efficientin deaminating A residues.

In some embodiments, the base editors provided herein comprise fusionproteins that further comprise one or more nuclear targeting sequences,for example, a nuclear localization sequence (NLS). In some embodiments,the fusion protein comprises multiple NLSs. In some embodiments, thefusion protein comprises a NLS at the N-terminus and the C-terminus ofthe fusion protein. In some embodiments, a NLS comprises an amino acidsequence that facilitates the importation of a protein, that comprisesan NLS, into the cell nucleus. In some embodiments, the NLS is fused tothe N-terminus of the fusion protein. In some embodiments, the NLS isfused to the C-terminus of the fusion protein. In some embodiments, theNLS is fused to the N-terminus of the programmable DNA binding protein,e.g. the Cas9. In some embodiments, the NLS is fused to the C-terminusof the programmable DNA binding protein. In some embodiments, the NLS isfused to the N-terminus of the adenosine deaminase. In some embodiments,the NLS is fused to the C-terminus of the adenosine deaminase. In someembodiments, the NLS is fused to the fusion protein via one or morelinkers. In some embodiments, the NLS is fused to the fusion proteinwithout a linker. In some embodiments, the NLS comprises an amino acidsequence of any one of the NLS sequences provided or referenced herein.In some embodiments, a NLS comprises the amino acid sequence PKKKRKV(SEQ ID NO: 27) or MD SLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 28).Additional nuclear localization sequences are known in the art and wouldbe apparent to the skilled artisan. For example, NLS sequences aredescribed in Plank et ah, PCT/EP2000/011690, the contents of which areincorporated herein by reference for their disclosure of exemplarynuclear localization sequences.

In some embodiments, the fusion proteins provided herein do not comprisea linker. In some embodiments, a linker is present between one or moreof the domains or proteins (e.g., adenosine deaminase, napDNAbp, NLS,and/or IBR). In some embodiments, the “-” used in the generalarchitecture above indicates the presence of an optional linker.

Some aspects of the disclosure provide base editors or fusion proteinsthat comprise a programmable DNA binding protein and at least twoadenosine deaminase domains. Without wishing to be bound by anyparticular theory, dimerization of adenosine deaminases (e.g., in cis orin trans) may improve the ability (e.g., efficiency) of the fusionprotein to modify a nucleic acid base, for example to deaminate adenine.In some embodiments, any of the fusion proteins may comprise 2, 3, 4 or5 adenosine deaminase domains. In some embodiments, any of the fusionproteins provided herein comprise two adenosine deaminases. In someembodiments, any of the fusion proteins provided herein contain only twoadenosine deaminases. In some embodiments, the adenosine deaminases arethe same. In some embodiments, the adenosine deaminases are any of theadenosine deaminases provided herein. In some embodiments, the adenosinedeaminases are different. In some embodiments, the first adenosinedeaminase is any of the adenosine deaminases provided herein, and thesecond adenosine is any of the adenosine deaminases provided herein, butis not identical to the first adenosine deaminase. In some embodiments,the first adenosine deaminase comprises any one of the mutationsprovided herein as numbered in SEQ ID NO: 1. In some embodiments, thesecond adenosine deaminase comprises any one of the mutations providedherein as numbered in SEQ ID NO: 1. In some embodiments, the firstadenosine deaminase comprises any one of the mutations provided hereinas numbered in SEQ ID NO: 1, and the second adenosine deaminasecomprises a wild type adenosine deaminase sequence. In some embodiments,the second adenosine deaminase comprises any one of the mutationsprovided herein as numbered in SEQ ID NO: 1, and the first adenosinedeaminase comprises a wild type adenosine deaminase sequence. As oneexample, the fusion protein may comprise a first adenosine deaminase anda second adenosine deaminase that both comprise a A106V, D108N, D147Y,and E155V mutation from ecTadA (SEQ ID NO: 1). As another example, thefusion protein may comprise a first adenosine deaminase domain thatcomprises a A106V, D108N, D147Y, and E155V mutation from ecTadA (SEQ IDNO: 1), and a second adenosine deaminase that comprises a L84F, A106V,D108N, H123Y, D147Y, E155V, and I156F mutation from ecTadA (SEQ ID NO:1).

In some embodiments, the adenosine deaminase comprises the amino acidsequence:

(SEQ ID NO: 1) MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTD

In some embodiments, the fusion protein comprises two adenosinedeaminases (e.g., a first adenosine deaminase and a second adenosinedeaminase). In some embodiments, the first adenosine deaminase isN-terminal to the second adenosine deaminase in the fusion protein. Insome embodiments, the first adenosine deaminase is C-terminal to thesecond adenosine deaminase in the fusion protein. In some embodiments,the first adenosine deaminase and the second deaminase are fuseddirectly or via a linker. In some embodiments, the linker is any of thelinkers provided herein, for example, any of the linkers described inthe “Linkers” section. In some embodiments, the first adenosinedeaminase is the same as the second adenosine deaminase. In someembodiments, the first adenosine deaminase and the second adenosinedeaminase are any of the adenosine deaminases described herein. In someembodiments, the first adenosine deaminase and the second adenosinedeaminase are different. In some embodiments, the first adenosinedeaminase is any of the adenosine deaminases provided herein. In someembodiments, the second adenosine deaminase is any of the adenosinedeaminases provided herein but is not identical to the first adenosinedeaminase. In some embodiments, the first adenosine deaminase is anecTadA adenosine deaminase. In some embodiments, the first adenosinedeaminase comprises an amino acid sequence that is at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to the amino acid sequence setforth SEQ ID NO: 1 or to any of the adenosine deaminases providedherein. In some embodiments, the second adenosine deaminase comprises anamino acid sequence that is at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%identical to the amino acid sequence set forth SEQ ID NO: 1 or to any ofthe adenosine deaminases provided herein. In some embodiments, thesecond adenosine deaminase comprises the amino acid sequence of SEQ IDNO: 1.

In some embodiments, the fusion proteins provided herein do not comprisea linker. In some embodiments, a linker is present between one or moreof the domains or proteins (e.g., first adenosine deaminase, secondadenosine deaminase, programmable DNA binding protein, and/or NLS).

It should be appreciated that the fusion proteins of the presentdisclosure may comprise one or more additional features. For example, insome embodiments, the fusion protein may comprise cytoplasmiclocalization sequences, export sequences, such as nuclear exportsequences, or other localization sequences, as well as sequence tagsthat are useful for solubilization, purification, or detection of thefusion proteins. Suitable protein tags provided herein include, but arenot limited to, biotin carboxylase carrier protein (BCCP) tags,myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags,polyhistidine tags, also referred to as histidine tags or His-tags,maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase(GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags,S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligasetags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequenceswill be apparent to those of skill in the art. In some embodiments, thefusion protein comprises one or more His tags.

In some embodiments, the fusion protein comprises an amino acid sequencethat is at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or atleast 99.9% identical to any one of the amino acid sequences listed inTable 23. In some embodiments, the fusion protein comprises any one ofthe amino acid sequences listed in Table 23. In some embodiments, thesequence of the fusion protein is any one of the amino acid sequenceslisted in Table 23. In some embodiments, the fusion protein comprises anamino acid sequence that is at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, atleast 99.7%, or at least 99.9% identical to any one of the amino acidsequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146,2152, 2156, and 2160. In some embodiments, the fusion protein comprisesany one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154,2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments,the sequence of the fusion protein is any one of the amino acidsequences of SEQ ID NOs: 2137, 2149, 2154, 2158, and 2188.

In some embodiments, the fusion protein is encoded by the polynucleotidesequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the polynucleotide sequenceslisted in Table 23. In some embodiments, the fusion protein is encodedby any one of the polynucleotide sequences listed in Table 23. In someembodiments, the fusion protein is expressed by the polynucleotidesequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the polynucleotide sequenceslisted in Table 23. In some embodiments, the fusion protein is expressedby any one of the polynucleotide sequences listed in Table 23. In someembodiments, the fusion protein is encoded by the polynucleotidesequence that comprises at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, atleast 99.7%, or at least 99.9% identical to any one of thepolynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161,2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162,2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187,and 2190. In some embodiments, the fusion protein is encoded by thepolynucleotide sequence that comprises any one of the polynucleotidesequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180,2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169,2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In someembodiments, the fusion protein is encoded by any one of thepolynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161,2168, 2174, 2180, 2186, and 2189. In some embodiments, the fusionprotein is expressed by the polynucleotide sequence that comprises atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.7%, or at least99.9% identical to any one of the polynucleotide sequences of SEQ IDNOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139,2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175,2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or acombination thereof: In some embodiments, the fusion protein is encodedby the polynucleotide sequence that comprises any one of thepolynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161,2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162,2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187,2190, 2138, 2147, 2158, or a combination thereof. In some embodiments,the fusion protein is expressed by any one of the polynucleotidesequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180,2186, and 2189. In some embodiments, the polynucleotide sequence furthercomprises at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the polynucleotide sequencesof SEQ ID NOs: 2138, 2147, 2158, or a combination thereof. In someembodiments, the polynucleotide sequence further comprises any one ofthe polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or acombination thereof.

In some embodiments, the nucleobase editor ABE8.8 comprises a fusionprotein comprising the sequence as provided below:

(SEQ ID NO: 3) MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGD

In some embodiments, the nucleobase editor comprises a fusion proteincomprising a polypeptide encoded by the polynucleotide (herein alsoreferenced as MA002) sequence as provided below:

(SEQ ID NO: 4) ATGAGCGAGGTCGAGTTCTCTCACGAATATTGGATGAGACACGCTCTCACCCTGGCTAAGAGAGCCAGGGACGAAAGAGAGGTGCCAGTTGGCGCTGTCCTGGTGTTGAACAATCGCGTCATCGGAGAAGGATGGAATCGCGCCATTGGCCTGCACGATCCAACCGCACATGCCGAAATTATGGCTCTGCGGCAAGGCGGCCTCGTGATGCAAAATTACAGACTGATCGATGCTACCCTCTACGTCACCTTCGAGCCCTGTGTCATGTGTGCTGGGGCAATGATTCACTCCCGGATTGGCCGCGTGGTGTTTGGAGTGCGGAATGCCAAGACTGGCGCCGCTGGATCTCTGATGGACGTCCTGCACcatCCTGGGATGAACCACCGGGTCGAGATCACAGAGGGAATTCTGGCTGACGAGTGCGCTGCCCTGCTGTGCaggTTCTTTAGAATGCCtAGAaggGTGTTCAACGCCCAGAAAAAAGCTCAGAGCAGCACCGATTCCGGCGGAAGCAGCGGAGGATCTTCTGGAAGCGAAACCCCAGGCACCAGCGAGTCTGCCACACCAGAATCATCTGGCGGTAGCTCCGGCGGCAGCGACAAGAAGTATTCTATCGGACTGGCCATCGGCACCAACTCTGTTGGATGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACAGGCACAGCATCAAGAAGAACCTGATCGGCGCACTGCTGTTCGACTCTGGCGAAACAGCCGAGGCCACCAGACTGAAGAGAACAGCCCGCAGACGGTACACCAGAAGAAAGAACCGGATCTGCTACCTCCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGACAAGAAGCACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGAGACTGATCTATCTGGCCCTGGCTCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAATCCTGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGAGTGGATGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCTGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACACCTAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAATCTGCTGGCCCAGATCGGCGATCAGTACGCCGACTTGTTTCTGGCCGCCAAGAATCTGAGCGACGCCATCCTGCTGTCCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCACCTCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGATCTGACCCTGCTGAAGGCCCTCGTTAGACAGCAGCTGCCAGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGATGGCGGAGCCAGCCAAGAGGAATTCTACAAGTTCATCAAGCCCATCCTCGAGAAGATGGACGGCACCGAGGAACTGCTGGTCAAGCTGAACAGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGCATCCCTCACCAGATCCACCTGGGAGAACTGCACGCCATTCTGCGGAGACAAGAGGACTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAAATCCTGACCTTCAGGATCCCCTACTACGTGGGACCACTGGCCAGAGGCAATAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACTCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCTCAGTCCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCTAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTTCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGATCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACAGCGTCGAGATCTCCGGCGTGGAAGATCGGTTCAATGCCAGCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAACGAAGAGAACGAGGACATCCTTGAGGACATCGTGCTGACACTGACCCTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACATACGCCCACCTGTTCGACGACAAAGTGATGAAGCAACTGAAGCGGCGGAGATACACCGGCTGGGGCAGACTGTCTCGGAAGCTGATCAACGGCATCCGGGATAAGCAGTCCGGCAAGACCATCCTGGACTTTCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATTCACGACGACAGCCTCACCTTCAAAGAGGATATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATTCTCTGCATGAGCACATTGCCAACCTGGCCGGCTCTCCCGCCATTAAGAAAGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTTGTGAAAGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACACAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGACGGGATATGTACGTGGACCAAGAGCTGGACATCAACAGACTGTCCGACTACGATGTGGACCATATCGTGCCCCAGTCTTTTCTGAAGGACGACTCCATCGACAACAAGGTCCTGACCAGATCCGACAAGAATCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTGGTCAAGAAGATGAAGAACTACTGGCGACAGCTGCTGAACGCCAAGCTGATTACCCAGCGGAAGTTCGACAATCTGACCAAGGCCGAAAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATTCTGGACTCTCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGCGAAGTGAAAGTCATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTCTACAAAGTGCGCGAGATCAACAACTACCATCACGCCCACGACGCCTACCTGAATGCCGTTGTTGGAACAGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAAGAGATTGGCAAGGCAACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACAGAGATCACCCTCGCCAACGGCGAGATCAGAAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGCGAGATTGTGTGGGATAAGGGCAGAGACTTTGCCACAGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAGAAAACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCTAAGCGGAACTCCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGATTCTCCTACCGTGGCCTATAGCGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTCAAGAGCGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCGATCGATTTCCTCGAGGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTCCCCAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCTGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCTAGCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCAGCCCCGAGGACAATGAGCAAAAGCAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTTAGCAAGAGAGTGATTCTGGCCGACGCCAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGACAAGCCTATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAACCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGCGGTACACCTCCACCAAAGAGGTGCTGGACGCCACTCTGATCCACCAGTCTATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAACTCGGAGGCGACGAAGGCGCCGATAAGAGAACCGCCGATGGCTCTGAGTTCGAGAGCCCCAAGA AAAAGCGCAAAGTG

In an aspect, a nucleobase editor system provided herein comprises aguide polynucleotide. In some embodiments, the guide polynucleotidebinds and forms a complex with the base editor fusion protein. In someembodiments, the guide polynucleotide directs the base editor fusionprotein to effect a modification at a target sequence. The guidepolynucleotide may comprise a single nucleic acid sequence or twoseparate nucleic acid sequences. In some embodiments, the guidepolynucleotide is a single guide, e.g. a single guide RNA. In someembodiments, the single guide RNA comprises a spacer sequence that iscapable of hybridizing with a target sequence. In some embodiments, thesingle guide RNA comprises a tracrRNA sequence that binds theprogrammable DNA binding protein, e.g. the Cas9 protein of the baseeditor fusion protein. In some embodiments, the single guide RNAcomprises a stem loop structure, a tracrRNA sequence, a crRNA sequence,a direct repeat, and/or an anti-repeat. In some embodiments, the singleguide RNA comprises a chemical modification. In some embodiments, theguide polynucleotide is any one of the guide polynucleotides providedherein, as described in the “Guide polynucleotide” section.

Deaminase Domains

Disclosed herein are base editor systems for editing, modifying oraltering a target nucleotide sequence of a polynucleotide.

A base editor system provided herein may comprise a programmable DNAbinding protein and a deaminase. As used herein, a deaminase may referto an enzyme that catalyzes the removal of an amine group from amolecule, or deamination, for example through hydrolysis. In someembodiments, the deaminase is a cytidine deaminase, catalyzing thedeamination of cytidine (C) to uridine (U), deoxycytidine (dC) todeoxyuridine (dU), or 5-methyl-cytidine to thymidine (T, 5-methyl-U),respectively. Subsequent DNA repair mechanisms ensure that a dU isreplaced by T, as described in Komor et al, Nature, Programmable editingof a target base in genomic DNA without double-stranded DNA cleavage,533, 420-424 (2016), which is incorporated herein by reference in itsentirety. In some embodiments, the deaminase is a cytosine deaminase,catalyzing and promoting the conversion of cytosine to uracil (e.g., inRNA) or thymine (e.g., in DNA). In some embodiments, the deaminase is anadenosine deaminase, catalyzing and promoting the conversion of adenineto guanine. In some embodiments, the deaminase is a naturally-occurringdeaminase from an organism, such as a human, chimpanzee, gorilla,monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is avariant of a naturally-occurring deaminase from an organism, and thevariants do not occur in nature. For example, in some embodiments, thedeaminase or deaminase domain is at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75% at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or at least 99.5% identical to a naturally-occurringdeaminase from an organism

A cytidine deaminase (or cytosine deaminase) comprises an enzyme thatcatalyzes the chemical reaction “cytosine+H20—>uracil+NH3” or“5-methyl-cytosine+H20—>thymine+NH3”. In the context of a gene, suchnucleotide change, or mutation, may in turn lead to an amino acid changein the protein, which may affect the protein's function, e.g.,loss-of-function or gain-of-function. Subsequent DNA repair mechanismsensure that uracil bases in DNA are replaced by T, as described in Komoret al. (Nature, Programmable editing of a target base in genomic DNAwithout double-stranded DNA cleavage, 533, 420-424 (2016), which isincorporated herein by reference in its entirety).

One exemplary suitable class of cytosine deaminases is theapolipoprotein B mRNA-editing complex (APOBEC) family of cytosinedeaminases encompassing eleven proteins that serve to initiatemutagenesis in a controlled and beneficial manner. The apolipoprotein Bediting complex 3 (APOBEC3) enzyme provides protection to human cellsagainst a certain HIV-1 strain via the deamination of cytosines inreverse-transcribed viral ssDNA. These cytosine deaminases all require aZn—coordinating motif (His-X-Glu-X23_26-Pro-Cys-X2_4-Cys (SEQ ID NO:29)) and bound water molecule for catalytic activity. The glutamic acidresidue acts to activate the water molecule to a zinc hydroxide fornucleophilic attack in the deamination reaction. Each family memberpreferentially deaminates at its own particular “hotspot,” for example,WRC (W is A or T, R is A or G) for hAID, or TTC for hAPOBEC3F. A recentcrystal structure of the catalytic domain of APOBEC3G revealed asecondary structure comprising a five-stranded β-sheet core flanked bysix α-helices, which is believed to be conserved across the entirefamily. The active center loops have been shown to be responsible forboth ssDNA binding and in determining “hotspot” identity. Overexpressionof these enzymes has been linked to genomic instability and cancer, thushighlighting the importance of sequence-specific targeting. Anothersuitable cytosine deaminase is the activation-induced cytidine deaminase(AID), which is responsible for the maturation of antibodies byconverting cytosines in ssDNA to uracils in a transcription-dependent,strand-biased fashion.

An adenosine deaminase (or adenine deaminase) comprises an enzyme thatcatalyzes the hydrolytic deamination of adenosine or deoxy adenosine toinosine or deoxyinosine, respectively. In some embodiments, theadenosine deaminase catalyzes the hydrolytic deamination of adenine oradenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g.engineered adenosine deaminases, evolved adenosine deaminases) providedherein may be from any organism, such as a bacterium. In someembodiments, the deaminase or deaminase domain is a variant of anaturally-occurring deaminase from an organism. In some embodiments, thedeaminase or deaminase domain does not occur in nature. For example, insome embodiments, the deaminase or deaminase domain is at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75% atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical to anaturally-occurring deaminase. In some embodiments, the adenosinedeaminase is from a bacterium, such as, E. coli, S. aureus, S. typhi, S.putrefaciens, H. influenzae, or C. crescentus. In some embodiments, theadenosine deaminase is a TadA deaminase. In some embodiments, the TadAdeaminase is an E. coli TadA deaminase. In some embodiments, the TadAdeaminase is a truncated E. coli TadA deaminase. For example, thetruncated ecTadA may be missing one or more N-terminal amino acidsrelative to a full-length ecTadA. In some embodiments, the truncatedecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the fulllength ecTadA. In some embodiments, the truncated ecTadA may be missing1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20C-terminal amino acid residues relative to the full length ecTadA. Insome embodiments, the ecTadA deaminase does not comprise an N-terminalmethionine.

In some embodiments, the adenosine deaminase comprises an amino acidsequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical tothe amino acid sequence set forth in SEQ ID NO:1 or to any of theadenosine deaminases provided herein. It should be appreciated thatadenosine deaminases provided herein may include one or more mutations(e.g., any of the mutations provided herein). The disclosure providesany deaminase domains with a certain percent identity plus any of themutations or combinations thereof described herein. In some embodiments,the adenosine deaminase comprises an amino acid sequence that has 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared tothe amino acid sequence set forth in SEQ ID NO:1 or any of the adenosinedeaminases provided herein. In some embodiments, the adenosine deaminasecomprises an amino acid sequence that has at least 5, at least 10, atleast 15, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 110, at least 120, at least 130, atleast 140, at least 150, at least 160, or at least 170 identicalcontiguous amino acid residues as compared to any one of the amino acidsequences set forth in SEQ ID NO:1 or any of the adenosine deaminasesprovided herein.

In some embodiments, the adenosine deaminase comprises a D108X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Ymutation in SEQ ID NO: 1, or a corresponding mutation in anotheradenosine deaminase. It should be appreciated, however, that additionaldeaminases may similarly be aligned to identify homologous amino acidresidues that can be mutated as provided herein.

In some embodiments, the adenosine deaminase comprises an A106X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an A106V mutation in SEQ ID NO: 1, ora corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises a E155X mutationin SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where the presence of X indicates any amino acid other thanthe corresponding amino acid in the wild-type adenosine deaminase. Insome embodiments, the adenosine deaminase comprises a E155D, E155G, orE155V mutation in SEQ ID NO: 1, or a corresponding mutation in anotheradenosine deaminase.

In some embodiments, the adenosine deaminase comprises a D147X mutationin SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where the presence of X indicates any amino acid other thanthe corresponding amino acid in the wild-type adenosine deaminase. Insome embodiments, the adenosine deaminase comprises a D147Y, mutation inSEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase.

It should be appreciated that any of the mutations provided herein(e.g., based on the ecTadA amino acid sequence of SEQ ID NO: 1) may beintroduced into other adenosine deaminases, such as S. aureus TadA(saTadA), or other adenosine deaminases (e.g., bacterial adenosinedeaminases). It would be apparent to the skilled artisan how to arehomologous to the mutated residues in ecTadA. Thus, any of the mutationsidentified in ecTadA may be made in other adenosine deaminases that havehomologous amino acid residues. It should also be appreciated that anyof the mutations provided herein may be made individually or in anycombination in ecTadA or another adenosine deaminase. For example, anadenosine deaminase may contain a D108N, a A106V, a E155V, and/or aD147Y mutation in ecTadA SEQ ID NO: 1, or a corresponding mutation inanother adenosine deaminase. In some embodiments, an adenosine deaminasecomprises the following group of mutations (groups of mutations areseparated by a “;”) in ecTadA SEQ ID NO: 1, or corresponding mutationsin another adenosine deaminase: D108N and A106V; D108N and E155V; D108Nand D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N,A106V, and E55V; D108N, A106V, and D147Y; D108N, E55V, and D147Y; A106V,E55V, and D 147Y; and D108N, A106V, E55V, and D147Y. It should beappreciated, however, that any combination of corresponding mutationsprovided herein may be made in an adenosine deaminase (e.g., ecTadA)

In some embodiments, the adenosine deaminase comprises one or more of aH8X, T17X, L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X,V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X,M151X, R153X, Q154X, I156X, and/or K157X mutation in SEQ ID NO: 1, orone or more corresponding mutations in another adenosine deaminase,where the presence of X indicates any amino acid other than thecorresponding amino acid in the wild-type adenosine deaminase. In someembodiments, the adenosine deaminase comprises one or more of H8Y, T17S,L18E, W23L, L34S, W45L, R51H, A56E, or A56S, E59G, E85K, or E85G, M94L,1951, V102A, F104L, A106V, R107C, or R107H, or R107P, D108G, or D108N,or D108V, or D108A, or D108Y, K1101, M118K, N127S, A138V, F149Y, M151V,R153C, Q154L, 1156D, and/or K157R mutation in SEQ ID NO: 1, or one ormore corresponding mutations in another adenosine deaminase. In someembodiments, the adenosine deaminase comprises one or more of themutations corresponding to SEQ ID NO: 1, or one or more correspondingmutations in another adenosine deaminase. In some embodiments, theadenosine deaminase comprises the mutation or mutations of in anyconstructs shown in Table 23 corresponding to SEQ ID NO: 1, or acorresponding mutation or mutations in another adenosine deaminase. Insome embodiments, the adenosine deaminase comprises one or more of aH8X, D108X, and/or N127X mutation in SEQ ID NO: 1, or one or morecorresponding mutations in another adenosine deaminase, where Xindicates the presence of any amino acid. In some embodiments, theadenosine deaminase comprises one or more of a H8Y, D108N, and/or N127Smutation in SEQ ID NO: 1, or one or more corresponding mutations inanother adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one or more ofH8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X,Q154X, E155X, K161X, Q163X, and/or T166X mutation in SEQ ID NO: 1, orone or more corresponding mutations in another adenosine deaminase,where X indicates the presence of any amino acid other than thecorresponding amino acid in the wild-type adenosine deaminase. In someembodiments, the adenosine deaminase comprises one or more of H8Y, R26W,M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H orQ154R, E155G or E155V or E155D, K161Q, Q163H, and/or T166P mutation inSEQ ID NO: 1, or one or more corresponding mutations in anotheradenosine deaminase.

In some embodiments, the adenosine deaminase comprises one, two, three,four, five, or six mutations selected from the group consisting of H8X,D108X, N127X, D147X, R152X, and Q154X in SEQ ID NO: 1, or acorresponding mutation or mutations in another adenosine deaminase,where X indicates the presence of any amino acid other than thecorresponding amino acid in the wild-type adenosine deaminase. In someembodiments, the adenosine deaminase comprises one, two, three, four,five, six, seven, or eight mutations selected from the group consistingof H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X in SEQ ID NO:1, or a corresponding mutation or mutations in another adenosinedeaminase, where X indicates the presence of any amino acid other thanthe corresponding amino acid in the wild-type adenosine deaminase. Insome embodiments, the adenosine deaminase comprises one, two, three,four, or five mutations selected from the group consisting of H8X,D108X, N127X, E155X, and T166X in SEQ ID NO: 1, or a correspondingmutation or mutations in another adenosine deaminase, where X indicatesthe presence of any amino acid other than the corresponding amino acidin the wild-type adenosine deaminase. In some embodiments, the adenosinedeaminase comprises one, two, three, four, five, or six mutationsselected from the group consisting of H8X, A106X, D108X, mutation ormutations in another adenosine deaminase, where X indicates the presenceof any amino acid other than the corresponding amino acid in thewild-type adenosine deaminase. In some embodiments, the adenosinedeaminase comprises one, two, three, four, five, six, seven, or eightmutations selected from the group consisting of H8X, R126X, L68X, D108X,N127X, D147X, and E155X in SEQ ID NO: 1, or a corresponding mutation ormutations in another adenosine deaminase, where X indicates the presenceof any amino acid other than the corresponding amino acid in thewild-type adenosine deaminase. In some embodiments, the adenosinedeaminase comprises one, two, three, four, or five mutations selectedfrom the group consisting of H8X, D108X, A109X, N127X, and E155X in SEQID NO: 1, or a corresponding mutation or mutations in another adenosinedeaminase, where X indicates the presence of any amino acid other thanthe corresponding amino acid in the wild-type adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one, two, three,four, five, or six mutations selected from the group consisting of H8Y,D108N, N127S, D147Y, R152C, and Q154H in SEQ ID NO: 1, or acorresponding mutation or mutations in another adenosine deaminase. Insome embodiments, the adenosine deaminase comprises one, two, three,four, five, six, seven, or eight mutations selected from the groupconsisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G and Q163H inSEQ ID NO: 1, or a corresponding mutation or mutations in anotheradenosine deaminase. In some embodiments, the adenosine deaminasecomprises one, two, three, four, or five mutations selected from thegroup consisting of H8Y, D108N, N127S, E155V, and T166P in SEQ ID NO: 1,or a corresponding mutation or mutations in another adenosine deaminase.In some embodiments, the adenosine deaminase comprises one, two, three,four, five, or six mutations selected from the group consisting of H8Y,A106T, D108N, N127S, E155D, and K161Q in SEQ ID NO: 1, or acorresponding mutation or mutations in another adenosine deaminase. Insome embodiments, the adenosine deaminase comprises one, two, three,four, five, six, seven, or eight mutations selected from the groupconsisting of H8Y, R126W, L68Q, D108N, N127S, D147Y, and E155V in SEQ IDNO: 1, or a corresponding mutation or mutations in another adenosinedeaminase. In some embodiments, the adenosine deaminase comprises one,two, three, four, or five mutations selected from the group consistingof H8Y, D108N, A109T, N127S, and E155G in SEQ ID NO: 1, or acorresponding mutation or mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises a D108N, D108G,or D108V mutation in SEQ ID NO: 1, or corresponding mutations in anotheradenosine deaminase. In some embodiments, the adenosine deaminasecomprises a A106V and D108N mutation in SEQ ID NO: 1, or correspondingmutations in another adenosine deaminase. In some embodiments, theadenosine deaminase comprises R107C and D108N mutations in SEQ ID NO: 1,or corresponding mutations in another adenosine deaminase. In someembodiments, the adenosine deaminase comprises a H8Y, D108N, N127S,D147Y, and Q154H mutation in SEQ ID NO: 1, or corresponding mutations inanother adenosine deaminase. In some embodiments, the adenosinedeaminase comprises a H8Y, R24W, D108N, N127S, D147Y, and E155V mutationin SEQ ID NO: 1, or corresponding mutations in another adenosinedeaminase. In some embodiments, the adenosine deaminase comprises aD108N, D147Y, and E155V mutation in SEQ ID NO: 1, or correspondingmutations in another adenosine deaminase. In some embodiments, theadenosine deaminase comprises a H8Y, D108N, and S 127S mutation in SEQID NO: 1, or corresponding mutations in another adenosine deaminase. Insome embodiments, the adenosine deaminase comprises a A106V, D108N,D147Y and E155V mutation in SEQ ID NO: 1, or corresponding mutations inanother adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one or more of aS2X, H8X, I49X, L84X, H123X, N127X, I156X and/or K160X mutation in SEQID NO: 1, or one or more corresponding mutations in another adenosinedeaminase, where the presence of X indicates any amino acid other thanthe corresponding amino acid in the wild-type adenosine deaminase. Insome embodiments, the adenosine deaminase comprises one or more of S2A,H8Y, I49F, L84F, H123Y, N127S, I156F and/or K160S mutation in SEQ ID NO:1, or one or more corresponding mutations in another adenosinedeaminase. In some embodiments, the adenosine deaminase comprises one ormore of the mutations in SEQ ID NO: 1, or one or more correspondingmutations in another adenosine deaminase. In some embodiments, theadenosine deaminase comprises the mutation or mutations of any one ofclones corresponding to SEQ ID NO: 1, or a corresponding mutation ormutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an L84X mutationadenosine deaminase, where X indicates any amino acid other than thecorresponding amino acid in the wild-type adenosine deaminase. In someembodiments, the adenosine deaminase comprises an L84F mutation in SEQID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an H123X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an H123Y mutation in SEQ ID NO: 1, ora corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an I157X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an I157F mutation in SEQ ID NO: 1, ora corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one, two, three,four, five, six, or seven mutations selected from the group consistingof L84X, A106X, D108X, H123X, D147X, E155X, and I156X in SEQ ID NO: 1,or a corresponding mutation or mutations in another adenosine deaminase,where X indicates the presence of any amino acid other than thecorresponding amino acid in the wild-type adenosine deaminase. In someembodiments, the adenosine deaminase comprises one, two, three, four,five, or six mutations selected from the group consisting of S2X, I49X,A106X, D108X, D147X, and E155X in SEQ ID NO: 1, or a correspondingmutation or mutations in another adenosine deaminase, where X indicatesthe presence of any amino acid other than the corresponding amino acidin the wild-type adenosine deaminase. In some embodiments, the adenosinedeaminase comprises one, two, three, four, or five mutations selectedfrom the group consisting of H8X, A106X, D108X, N127X, and K160X in SEQID NO: 1, or a corresponding mutation or mutations in another adenosinedeaminase, where X indicates the presence of any amino acid other thanthe corresponding amino acid in the wild-type adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one, two, three,four, five, six, or seven mutations selected from the group consistingof L84F, A106V, D108N, H123Y, D147Y, E155V, and 1156F in SEQ ID NO: 1,or a corresponding mutation or mutations in another adenosine deaminase.In some embodiments, the adenosine deaminase comprises one, two, three,four, five, or six mutations selected from the group consisting of S2A,I49F, A106V, D108N, D147Y, and E155V in SEQ ID NO: 1, or a correspondingamino acid in another adenosine deaminase. In some embodiments, theadenosine deaminase comprises one, two, three, four, or five mutationsselected from the group consisting of H8Y, A106T, D108N, N127S, andK160S in SEQ ID NO: 1, or a corresponding mutation or mutations inanother adenosine deaminase. In some embodiments, the adenosinedeaminase comprises one or more of a E25X, R26X, R107X, A142X, and/orA143X mutation in SEQ ID NO: 1, or one or more corresponding mutationsin another adenosine deaminase, where the presence of X indicates anyamino acid other than the corresponding amino acid in the wild-typeadenosine deaminase. In some embodiments, the adenosine deaminasecomprises one or more of E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G,R26N, R26Q, R26C, R26L, R26K, R107P, R07K, R107A, R107N, R107W, R107H,R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M,A143S, A143Q and/or A143R mutation in SEQ ID NO: 1, or one or morecorresponding mutations in another adenosine deaminase. In someembodiments, the adenosine deaminase comprises one or more of themutations provided in Table 23 corresponding to SEQ ID NO: 1, or one ormore corresponding mutations in another adenosine deaminase. In someembodiments, the adenosine deaminase comprises the mutation or mutationsof any one shown in Table 23 corresponding to SEQ ID NO: 1, or acorresponding mutation or mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an E25X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S,or E25Y mutation in SEQ ID NO: 1, or a corresponding mutation in anotheradenosine deaminase.

In some embodiments, the adenosine deaminase comprises an R26X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an, R26G, R26N, R26Q, R26C, R26L, orR26K mutation in SEQ ID NO: 1, or a corresponding mutation in anotheradenosine deaminase.

In some embodiments, the adenosine deaminase comprises an R107X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an R107P, R07K, R107A, R107N, R107W,R107H, or R107S mutation in SEQ ID NO: 1, or a corresponding mutation inanother adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an A142X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an A142N, A142D, A142G, mutation inSEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase.

In some embodiments, the adenosine deaminase comprises an A143X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W,A143M, A143S, A143Q and/or A143R mutation in SEQ ID NO: 1, or acorresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one or more of aH36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S 146X, Q154X,K157X, and/or K161X mutation in SEQ ID NO: 1, or one or morecorresponding mutations in another adenosine deaminase, where thepresence of X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises one or more of H36L, N37T, N37S, P48T,P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S 146R, S 146C, Q154H,K157N, and/or K161T mutation in SEQ ID NO: 1, or one or morecorresponding mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an H36X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an H36L mutation in SEQ ID NO: 1, or acorresponding mutation in another adenosine deaminase. In someembodiments, the adenosine deaminase comprises an N37X mutation inecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an N37T, or N37S mutation in SEQ IDNO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an P48X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an P48T, or P48L mutation in SEQ IDNO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an R51X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises an R51H, or R51L mutation in SEQ IDNO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an S 146Xmutation in ecTadA SEQ ID NO: 1, or a corresponding mutation in anotheradenosine deaminase, where X indicates any amino acid other than thecorresponding amino acid in the wild-type adenosine deaminase. In someembodiments, the adenosine deaminase comprises an S 146R, or S 146Cmutation in SEQ ID NO: 1, or a corresponding mutation in anotheradenosine deaminase.

In some embodiments, the adenosine deaminase comprises an K157X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises a K157N mutation in SEQ ID NO: 1, or acorresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an P48X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises a P48S, P48T, or P48A mutation in SEQID NO: 1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an A142X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises a A142N mutation in SEQ ID NO: 1, or acorresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an W23X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises a W23R, or W23L mutation in SEQ ID NO:1, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an R152X mutationin ecTadA SEQ ID NO: 1, or a corresponding mutation in another adenosinedeaminase, where X indicates any amino acid other than the correspondingamino acid in the wild-type adenosine deaminase. In some embodiments,the adenosine deaminase comprises a R152P, or R52H mutation in SEQ IDNO: 1, or a corresponding mutation in another adenosine deaminase.

Additional adenosine deaminase mutations and variants are described inPatent Application WO2018119354, which is incorporated herein byreference in its entirety.

Additional adenosine deaminases useful in the present application wouldbe apparent to the skilled artisan and are within the scope of thisdisclosure. For example, the adenosine deaminase may be a homolog of anAD AT. Exemplary AD AT homologs include, without limitation:

Staphylococcus aureus TadA: (SEQ ID NO: 30)MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKN LRANKKSTNBacillus subtilis TadA: (SEQ ID NO: 31)MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRK KKKAARKNLSESalmonella typhimurium (S. typhimurium) TadA: (SEQ ID NO: 32)MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAVShewanella putrefaciens (S. putrefaciens) TadA: (SEQ ID NO: 33)MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEK KALKLAQRAQQGIEHaemophilus influenzae F3031 (H. influenzae) TadA: (SEQ ID NO: 34)MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK Caulobacter crescentus (C. crescentus) TadA:(SEQ ID NO: 35) MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLR GFFRARRKAKIGeobacter sulfurreducens (G. sulfurreducens) TadA: (SEQ ID NO: 36)MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

In some embodiments, the adenosine deaminase comprises an amino acidsequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the amino acid sequenceslisted in Table 23. In some embodiments, the adenosine deaminasecomprises any one of the amino acid sequences listed in Table 23. Insome embodiments, the sequence of the adenosine deaminase is any one ofthe amino acid sequences listed in Table 23. In some embodiments, theadenosine deaminase comprises an amino acid sequence that is at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9%identical to any one of the amino acid sequences of SEQ ID NOs: SEQ IDNOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160.In some embodiments, the adenosine deaminase comprises any one of theamino acid sequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140,40, 2146, 2152, 2156, and 2160. In some embodiments, the sequence of theadenosine deaminase is any one of the amino acid sequences of SEQ IDNOs: 2137, 2149, 2154, 2158, and 2188.

In some embodiments, the adenosine deaminase is encoded by thepolynucleotide sequence that is at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, at least 99.7%, or at least 99.9% identical to any one of thepolynucleotide sequences listed in Table 23. In some embodiments, theadenosine deaminase is encoded by any one of the polynucleotidesequences listed in Table 23. In some embodiments, the adenosinedeaminase is expressed by the polynucleotide sequence that is at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, at least 99.7%, or at least 99.9%identical to any one of the polynucleotide sequences listed in Table 23.In some embodiments, the adenosine deaminase is expressed by any one ofthe polynucleotide sequences listed in Table 23. In some embodiments,the adenosine deaminase is encoded by the polynucleotide sequence thatcomprises at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the polynucleotide sequencesof SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186,2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171,2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In someembodiments, the adenosine deaminase is encoded by the polynucleotidesequence that comprises any one of the polynucleotide sequences of SEQID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189,2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173,2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In some embodiments,the adenosine deaminase is encoded by any one of the polynucleotidesequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180,2186, and 2189. In some embodiments, the adenosine deaminase isexpressed by the polynucleotide sequence that comprises at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, at least 99.7%, or at least 99.9% identicalto any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148,2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151,2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181,2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof: Insome embodiments, the adenosine deaminase is encoded by thepolynucleotide sequence that comprises any one of the polynucleotidesequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180,2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169,2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147,2158, or a combination thereof. In some embodiments, the adenosinedeaminase is expressed by any one of the polynucleotide sequences of SEQID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189.In some embodiments, the polynucleotide sequence further comprises atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.7%, or at least99.9% identical to any one of the polynucleotide sequences of SEQ IDNOs: 2138, 2147, 2158, or a combination thereof. In some embodiments,the polynucleotide sequence further comprises any one of thepolynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, or acombination thereof.

Programmable DNA Binding Proteins

Provided herein are programmable DNA-binding proteins that can beprogrammed to target to bind DNA sequences in any desired nucleotidesequence within a genome. To program the DNA-binding protein to bind adesired nucleotide sequence, the DNA binding protein may be modified tochange its binding specificity, e.g., zinc finger DNA-binding domain,zinc finger nuclease (ZFN), a CRISPR-Cas9 protein, or a transcriptionactivator—like effector proteins (TALE). ZFNs are artificial restrictionenzymes generated by fusing a zinc finger DNA-binding domain to aDNA-cleavage domain. Zinc finger domains can be engineered to targetspecific desired DNA sequences and this enables zinc-fingers to bindunique sequences within complex genomes. Transcription activator-likeeffector nucleases (TALEN) are engineered restriction enzymes that canbe engineered to cut specific sequences of DNA. They are made by fusinga TAL effector DNA-binding domain to a nuclease domain (e.g. Fokl).Transcription activator-like effectors (TALEs) can be engineered to bindpractically any desired DNA sequence. Methods for programming ZFNs andTALEs are familiar to one skilled in the art. For example, such methodsare described in Maeder, et al, Mol. Cell 31 (2): 294-301, 2008; Carrollet al, Genetics Society of America, 188 (4): 773-782, 2011; Miller etal., Nature Biotechnology 25 (7): 778-785, 2007; Christian et al,Genetics 186 (2): 757-61, 2008; Li et al, Nucleic Acids Res. 39 (1):359-372, 2010; and Moscou et al, Science 326 (5959): 1501, 2009, each ofwhich are incorporated herein by reference.

A CRISPR/Cas system or a Cas protein in a base editor system providedherein may comprise Class 1 or Class 2 system components, includingribonucleic acid protein complexes. The Class 2 Cas nuclease families ofproteins are enzymes with DNA endonuclease activity, and they can bedirected to cleave a desired nucleic acid target by designing anappropriate guide RNA, as described further herein. A Class 2 CRISPR/Cassystem component may be from a Type II, Type IIA, Type IIB, Type IIC,Type V, or Type VI system. Class 2 Cas nucleases include, for example,Cas9 (also known as Csn1 or Csx12), Csn2, Cas4, Cas12a (Cpf1), Cas12b(C2c1), Cas12c (C2c3), Cas13a (C2c2), Cas13b, Cas13c, and Cas13dproteins. In some embodiments, the Cas protein is from a Type IICRISPR/Cas system, i.e., a Cas9 protein from a CRISPR/Cas9 system, or aType V CRISPR/Cas system, e.g., a Cas12a protein. In some embodiments,the Cas protein is from a Class 2 CRISPR/Cas system, i.e., asingle-protein Cas nuclease such as a Cas9 protein or a Cas12a protein.

Other non-limiting examples of Cas proteins can include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1,Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3,Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX,Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, Cas9HiFi, homologuesthereof, or modified versions thereof.

In some embodiments, provided herein are guide nucleotidesequence-programmable DNA-binding protein or RNA guided programable DNAbinding proteins that are able to bind DNA, and the binding to itstarget DNA sequence is mediated by a guide nucleotide sequence. Thus, itis appreciated that the guide nucleotide sequence-programmableDNA-binding protein binds to a guide nucleotide sequence. The guidenucleotide may be an RNA or DNA molecule (e.g., a single-stranded DNA orssDNA molecule) that is complementary to the target sequence and canguide the DNA binding protein to the target sequence. As such, a guidenucleotide sequence-programmable DNA-binding protein may be aRNA-programmable DNA-binding protein (e.g., a Cas9 protein), or anssDNA-programmable DNA-binding protein (e.g., an Argonaute protein).“Programmable” means the DNA-binding protein may be programmed to bindany DNA sequence that the guide nucleotide targets. Exemplary guidenucleotide sequence-programmable DNA-binding proteins include, but arenot limited to, Cas9 (e.g., dCas9 and nCas9), saCas9 (e.g., saCas9d,saCas9d, saKKH Cas9) CasX, CasY, Cpf1, C2c1, C2c2, C2c3, Argonaute, andany other suitable protein described herein, or variants thereof.

In some embodiments, the guide nucleotide sequence exists as a singlenucleotide molecule and comprises two domains: (1) a domain that shareshomology to a target nucleic acid (e.g., and directs binding of a guidenucleotide sequence-programmable DNA-binding protein to the target); and(2) a domain that binds a guide nucleotide sequence-programmableDNA-binding protein. In some embodiments, domain (1) comprises a spacersequence. In some embodiments, domain (2) is referred to as a tracrRNAsequence. In some embodiments, domain (2) corresponds to a sequenceknown as a tracrRNA, and comprises a stem-loop structure. For example,in some embodiments, domain (2) is identical or homologous to a tracrRNAas provided in Jinek et al., Science 337:816-821(2012), which isincorporated herein by reference. Other examples of gRNAs (e.g., thoseincluding domain 2) can be found in U.S. Patent Application PublicationUS20160208288 and U.S. Patent Application Publication US20160200779 eachof which is herein incorporated by reference in their entirety.

Methods of using guide nucleotide sequence-programmable DNA-bindingprotein, such as Cas9, for site-specific cleavage (e.g., to modify agenome) are known in the art (see e.g., Cong, L. et al. Multiplex genomeengineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali,P. et al. RNA-guided human genome engineering via Cas9. Science 339,823-826 (2013); Hwang, W. Y. et al. Efficient genome editing inzebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229(2013); Jinek, M. et al. RNA-programmed genome editing in human cells.eLife 2, e00471 (2013); Dicarlo, J. E. et al. Genome engineering inSaccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acidsresearch (2013); Jiang, W. et al. RNA-guided editing of bacterialgenomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239(2013); each of which are incorporated herein by reference).

CRISPR is an adaptive immune system that provides protection againstmobile genetic elements (viruses, transposable elements and conjugativeplasmids). CRISPR clusters contain spacers, sequences complementary toantecedent mobile elements, and target invading nucleic acids. ACRISPR/Cas system comprises a non-coding RNA molecule (e.g., guide RNA)that binds to DNA (e.g., target DNA sequence) and Cas proteins (e.g.,Cas9) with nuclease functionality (e.g., two nuclease domains). See,e.g., Sander, et al., Nature Biotechnology, 32:347-355 (2014); see alsoe.g., Hsu, et al., Cell 157(6):1262-1278 (2014). The general mechanismand recent advances of CRISPR system is discussed in Cong, et al.,Science, 339(6121): 819-823 (2013); Fu, et al., Nature Biotechnology,31, 822-826 (2013); Chu, et al., Nature Biotechnology 33, 543-548(2015); Shmakov, et al., Molecular Cell, 60, 1-13 (2015); Makarova, etal., Nature Reviews Microbiology, 13, 1-15 (2015). CRISPR/Cas systemscan be used to introduce site-specific cleavage of a target DNA. Thelocations for site-specific cleavage are determined by both 1)base-pairing complementarity between the guide RNA (gRNA) and the targetDNA (a protospacer) and 2) a short motif in the target DNA referred toas the protospacer adjacent motif (PAM). CRISPR/Cas systems (e.g., TypeII CRISPR/Cas system) can be used to generate, e.g., an engineered cellin which a target gene is disrupted or mutated. A Cas enzyme (e.g.,Cas9) can be used to catalyze DNA cleavage. A Cas9 protein (e.g., aStreptococcus pyogenes Cas9 or any closely related Cas9) can derive anenzymatic action to generate double stranded breaks at target sitesequences which hybridize to about 20 nucleotides of a guide sequence(e.g., gRNA) and that have a protospacer-adjacent motif (PAM) followingthe target sequence.

CRISPR/Cas system comprises Class 1 or Class 2 system components,including ribonucleic acid protein complexes. The Class 2 Cas nucleasefamilies of proteins are enzymes with DNA endonuclease activity, andthey can be directed to cleave a desired nucleic acid target bydesigning an appropriate guide RNA, as described further herein. A Class2 CRISPR/Cas system component may be from a Type II, Type IIA, Type IIB,Type IIC, Type V, or Type VI system. Class 2 Cas nucleases include, forexample, Cas9 (also known as Csn1 or Csx12), Csn2, Cas4, Cas12a (Cpf1),Cas12b (C2c1), Cas12c (C2c3), Cas13a (C2c2), Cas13b, Cas13c, and Cas13dproteins. In some embodiments, the Cas protein is from a Type IICRISPR/Cas system, i.e., a Cas9 protein from a CRISPR/Cas9 system, or aType V CRISPR/Cas system, e.g., a Cas12a protein. In some embodiments,the Cas protein is from a Class 2 CRISPR/Cas system, i.e., asingle-protein Cas nuclease such as a Cas9 protein or a Cas12a protein.

Other non-limiting examples of Cas proteins can include Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1,Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3,Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX,Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, Cas9HiFi, homologuesthereof, or modified versions thereof.

A base editor system provided herein may comprise a Cas9 or a Cas9nuclease. A Cas9 protein or a Cas9 nuclease refers to an RNA-guidednuclease comprising a Cas9 protein, a fragment, or a variant thereof. ACas9 nuclease is also referred to sometimes as a casn1 nuclease or aCRISPR (clustered regularly interspaced short palindromicrepeat)—associated nuclease. CRISPR is an adaptive immune system thatprovides protection against mobile genetic elements (viruses,transposable elements and conjugative plasmids). CRISPR clusters containspacers, sequences complementary to antecedent mobile elements, andtarget invading nucleic acids. CRISPR clusters are transcribed andprocessed into CRISPR RNA (crRNA). In type II CRISPR systems correctprocessing of pre-crRNA requires a trans-encoded small RNA (tracrRNA),endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA servesas a guide for ribonuclease 3-aided processing of pre-crRNA.Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear orcircular dsDNA target complementary to the spacer. The target strand notcomplementary to crRNA is first cut endonucleolytically, then trimmed3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typicallyrequires protein and both RNAs. However, single guide RNAs (“sgRNA”, orsimply “gRNA”) can be engineered so as to incorporate aspects of boththe crRNA and tracrRNA into a single RNA species. See, e.g., Jinek etal., Science 337:816-821(2012), which is incorporated herein byreference in its entirety.

The term “Cas9” refers to an RNA guided nuclease comprising a Cas9protein, or a fragment thereof (e.g., a protein comprising an active,inactive, or partially active DNA cleavage domain of Cas9, and/or thegRNA binding domain of Cas9). A Cas9 nuclease is also referred to as aCasn1 nuclease or a CRISPR (clustered regularly interspaced shortpalindromic repeat) associated nuclease. Cas9 can refer to a polypeptidewith at least or at least about 50%, 60%, 70%, 80%, 90%, 100% sequenceidentity and/or sequence similarity to a wild type exemplary Cas9polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to apolypeptide with at most or at most about 50%, 60%, 70%, 80%, 90%, 100%sequence identity and/or sequence similarity to a wild type exemplaryCas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to the wildtype or a modified form of the Cas9 protein that can comprise an aminoacid change such as a deletion, insertion, substitution, variant,mutation, fusion, chimera, or any combination thereof.

Cas9 nuclease sequences and structures of variant Cas9 orthologs havebeen described in various species. Exemplary species that the Cas9protein or other components can be from include, but are not limited to,Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.,Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri,Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis,Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni,Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum,Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomycesviridochromogenes, Streptomyces viridochromogenes, Streptosporangiumroseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides,Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillusdelbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponemadenticola, Microscilla marina, Burkholderiales bacterium, Polar omonasnaphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothecesp., Microcystis aeruginosa, Synechococcus sp., Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, CandidatusDesulforudis, Clostridium botulinum, Clostridium difficile, Finegoldiamagna, Natranaerobius thermophilus, Pelotomaculum thermopropionium,Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatiumvinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcuswatsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer,Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena,Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp.,Lyngbya sp., Microcoleus chthonoplastes, Oscillator ia sp., Petrotogamobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseriacinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, or Acaryochloris marina. In some embodiments, theCas9 protein is from Streptococcus pyogenes. In some embodiments, theCas9 protein may be from Streptococcus thermophilus. In someembodiments, the Cas9 protein is from Staphylococcus aureus.

Additional suitable Cas9 nucleases and sequences will be apparent tothose of skill in the art based on this disclosure, and such Cas9nucleases and sequences include Cas9 sequences from the organisms andloci disclosed in Chylinski et al., (2013) RNA Biology 10:5, 726-737;which are incorporated herein by reference.

In some embodiments, wild-type Cas9 corresponds to Cas9 fromStreptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotidesequence as follows); and Uniprot Reference Sequence: 99ZW2 amino acidsequence as follows

(SEQ ID NO: 37) ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGG TGACTGA(SEQ ID NO: 38) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

In some embodiments, Cas9 is a Cas9 protein from species Corynebacteriumulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacteriumdiphtheria (NCBI Refs. NC 016782.1, NC_016786.1); Spiroplasmasyrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref:NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1);Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBIRef: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC_018721.1);Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBIRef: YP_002344900.1) or Neisseria, meningitidis (NCBI Ref:YP_002342100.1).

In some embodiments, a base editor provided herein comprises aprogrammable DNA binding protein, e.g. a Cas nuclease, with reduced orabolished nuclease activity. For example a Ca9 protein may be nucleaseinactive or may be a Cas9 nickase. Methods for generating a Cas9 protein(or a fragment thereof) having an inactive DNA cleavage domain are known(See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al, (2013)Cell. 28; 152(5): 1173-83, each of which are incorporated herein byreference in its entirety. For example, the DNA cleavage domain of Cas9is known to include two subdomains, the HNH nuclease subdomain and theRuvC1 subdomain. The HNH subdomain cleaves the strand complementary tothe gRNA, whereas the RuvC1 subdomain cleaves the non-complementarystrand. Mutations within these subdomains can silence the nucleaseactivity of Cas9. For example, the mutations D10A and H840A completelyinactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al.,Science. 337:816-821(2012); Qi et al, Cell. 28;152(5): 1173-83 (2013)).The Cas9 nickase suitable for use in accordance with the presentdisclosure has an active HNH domain and an inactive RuvC domain and isable to cleave only the strand of the target DNA that is bound by thesgRNA (which is the opposite strand of the strand that is being editedvia cytidine deamination). The Cas9 nickase of the present disclosuremay comprise mutations that inactivate the RuvC domain, e.g., a D10Amutation. It is to be understood that any mutation that inactivates theRuvC domain may be included in a Cas9 nickase, e.g., insertion,deletion, or single or multiple amino acid substitution in the RuvCdomain. In a Cas9 nickase described herein, while the RuvC domain isinactivated, the HNH domain remains activate. Thus, while the Cas9nickase may comprise mutations other than those that inactivate the RuvCdomain (e.g., D10A), those mutations do not affect the activity of theHNH domain. In a non-limiting Cas9 nickase example, the histidine atposition 840 remains unchanged.

In some embodiments, a nuclease inactive Cas9 comprises the amino acidsequence of dCas9 (D10A and H840A) provided below:

(SEQ ID NO: 39) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

In some embodiments, a Cas9 nickase comprises the amino acid sequence ofan exemplary catalytically Cas9 nickase (nCas9) is as follows:

(SEQ ID NO: 40) DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS ITGLYETRIDLSQLGGD

Additional suitable mutations that inactivate Cas9 will be apparent tothose of skill in the art based on this disclosure and knowledge in thefield, and are within the scope of this disclosure. Such additionalexemplary suitable nuclease-inactive Cas9 domains include, but are notlimited to, D839A and/or N863A (See, e.g., Prashant et al, NatureBiotechnology. 2013; 31(9): 833-838, which are incorporated herein byreference), or), or K603R {See, e.g., Chavez et al., Nature Methods 12,326-328, 2015, which is incorporated herein by reference). Cas9, dCas9,or Cas9 variant also encompasses Cas9, dCas9, or Cas9 variants from anyorganism. Also appreciated is that dCas9, Cas9 nickase, or otherappropriate Cas9 variants from any organisms may be used in accordancewith the present disclosure.

In some embodiments, Cas9 refers to a Cas9 from archaea (e.g.nanoarchaea), which constitute a domain and kingdom of single-celledprokaryotic microbes.

In some embodiments, the programmable DNA binding protein comprises aCasX or CasY, or a variant thereof, which have been described in, forexample, Burstein et al., “New CRISPR-Cas systems from uncultivatedmicrobes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entirecontents of which is hereby incorporated by reference. Usinggenome-resolved metagenomics, a number of CRISPR-Cas systems wereidentified, including the first reported Cas9 in the archaeal domain oflife. This divergent Cas9 protein was found in little-studiednanoarchaea as part of an active CRISPR-Cas system. In bacteria, twopreviously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY,which are among the most compact systems yet discovered.

Some aspects of the disclosure provide high fidelity Cas9 domains of thenucleobase editors provided herein. In some embodiments, high fidelityCas9 domains are engineered Cas9 domains comprising one or moremutations that decrease electrostatic interactions between the Cas9domain and the sugar-phosphate backbone of DNA, as compared to acorresponding wild-type Cas9 domain. Without wishing to be bound by anyparticular theory, high fidelity Cas9 domains that have decreasedelectrostatic interactions with the sugar-phosphate backbone of DNA mayhave less off-target effects. In some embodiments, the Cas9 domaincomprises one or more mutations that decreases the association betweenthe Cas9 domain and the sugar-phosphate backbone of DNA. In someembodiments, a Cas9 domain comprises one or more mutations thatdecreases the association between the Cas9 domain and thesugar-phosphate backbone of DNA by at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, ormore. In some embodiments, any of the Cas9 fusion proteins providedherein comprise one or more of N497X, R661X, Q695X, and/or Q926Xmutation as numbered in the wild type Cas9 amino acid sequence or acorresponding amino acid in another Cas9, wherein X is any amino acid.In some embodiments, any of the Cas9 or Cas9 fusion proteins providedherein comprise one or more of N497A, R661A, Q695A, and/or Q926Amutation of the amino acid sequence provided in the wild type Cas9sequence, or a corresponding mutation as numbered in the wild type Cas9amino acid sequence or a corresponding amino acid in another Cas9. Cas9domains with high fidelity have been described in Kleinstiver, B. P., etal. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wideoff-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I. M.,et al. “Rationally engineered Cas9 nucleases with improved specificity.”Science 351, 84-88 (2015); the entire contents of each are incorporatedherein by reference.

It should be appreciated that any of the base editors provided herein,for example, any of the adenosine deaminase base editors providedherein, may be converted into high fidelity base editors by modifyingthe Cas9 domain as described herein to generate high fidelity baseeditors, for example, a high fidelity adenosine base editor. In someembodiments, the high fidelity Cas9 domain is a nuclease inactive Cas9domain. In some embodiments, the high fidelity Cas9 domain is a Cas9nickase domain.

In some embodiments, a Cas protein comprises a CasX or CasY, or avariant thereof, which have been described in, for example, Burstein etal., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is herebyincorporated by reference. Using genome-resolved metagenomics, a numberof CRISPR-Cas systems were identified, including the first reported Cas9in the archaeal domain of life. This divergent Cas9 protein was found inlittle-studied nanoarchaea as part of an active CRISPR-Cas system. Inbacteria, two previously unknown systems were discovered, CRISPR-CasXand CRISPR-CasY, which are among the most compact systems yetdiscovered.

Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleasesfrom the Cpf1 family that exhibit cleavage activity in mammalian cells.Cpf1-mediated DNA cleavage is a double-strand break with a short 3′overhang, which is different from Cas9-mediated DNA cleavage. Thestaggered cleavage pattern of Cpf1 may open up the possibility ofdirectional gene transfer, analogous to traditional restriction enzymecloning, which may increase the efficiency of gene editing. Like theCas9 variants and orthologues described above, Cpf1 may also expand thenumber of sites that can be targeted by CRISPR to AT-rich regions orAT-rich genomes that lack the NGG PAM sites favored by SpCas9.

In some embodiments, the Cas9 protein comprises an amino acid sequencethat is at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or atleast 99.9% identical to any one of the amino acid sequences listed inTable 23. In some embodiments, the Cas9 protein comprises any one of theamino acid sequences listed in Table 23. In some embodiments, thesequence of the Cas9 protein is any one of the amino acid sequenceslisted in Table 23. In some embodiments, the Cas9 protein comprises anamino acid sequence that is at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, atleast 99.7%, or at least 99.9% identical to any one of the amino acidsequences of SEQ ID NOs: 2137, 2149, 2154, 2158, 2188, 2140, 40, 2146,2152, 2156, and 2160. In some embodiments, the Cas9 protein comprisesany one of the amino acid sequences of SEQ ID NOs: 2137, 2149, 2154,2158, 2188, 2140, 40, 2146, 2152, 2156, and 2160. In some embodiments,the sequence of the Cas9 protein is any one of the amino acid sequencesof SEQ ID NOs: 2137, 2149, 2154, 2158, and 2188.

In some embodiments, the Cas9 protein is encoded by the polynucleotidesequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the polynucleotide sequenceslisted in Table 23. In some embodiments, the Cas9 protein is encoded byany one of the polynucleotide sequences listed in Table 23. In someembodiments, the Cas9 protein is expressed by the polynucleotidesequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the polynucleotide sequenceslisted in Table 23. In some embodiments, the Cas9 protein is expressedby any one of the polynucleotide sequences listed in Table 23. In someembodiments, the Cas9 protein is encoded by the polynucleotide sequencethat is at In some embodiments, the Cas9 protein is encoded by thepolynucleotide sequence that comprises at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, at least 99.7%, or at least 99.9% identical to any one ofthe polynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157,2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159,2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185,2187, and 2190. In some embodiments, the Cas9 protein is encoded by thepolynucleotide sequence that comprises any one of the polynucleotidesequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180,2186, 2189, 2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169,2171, 2173, 2175, 2177, 2179, 2181, 2183, 2185, 2187, and 2190. In someembodiments, the Cas9 protein is encoded by any one of thepolynucleotide sequences of SEQ ID NOs: 2192, 2148, 2153, 2157, 2161,2168, 2174, 2180, 2186, and 2189. In some embodiments, the Cas9 proteinis expressed by the polynucleotide sequence that comprises at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, at least 99.7%, or at least 99.9% identicalto any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148,2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189, 2139, 2142, 2145, 2151,2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173, 2175, 2177, 2179, 2181,2183, 2185, 2187, 2190, 2138, 2147, 2158, or a combination thereof: Insome embodiments, the Cas9 protein is encoded by the polynucleotidesequence that comprises any one of the polynucleotide sequences of SEQID NOs: 2192, 2148, 2153, 2157, 2161, 2168, 2174, 2180, 2186, 2189,2139, 2142, 2145, 2151, 2155, 2159, 2162, 2164, 2167, 2169, 2171, 2173,2175, 2177, 2179, 2181, 2183, 2185, 2187, 2190, 2138, 2147, 2158, or acombination thereof. In some embodiments, the Cas9 protein is expressedby any one of the polynucleotide sequences of SEQ ID NOs: 2192, 2148,2153, 2157, 2161, 2168, 2174, 2180, 2186, and 2189. In some embodiments,the polynucleotide sequence further comprises at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, at least 99.7%, or at least 99.9% identical to anyone of the polynucleotide sequences of SEQ ID NOs: 2138, 2147, 2158, ora combination thereof. In some embodiments, the polynucleotide sequencefurther comprises any one of the polynucleotide sequences of SEQ ID NOs:2138, 2147, 2158, or a combination thereof.

Linkers

Base editors provided herein may comprises linkers that connect one ormore components of the base editors. In certain embodiments, linkers maybe used to link any of the protein or protein domains described herein.The linker may be as simple as a covalent bond, or it may be a polymericlinker many atoms in length. In certain embodiments, the linker is apolypeptide or based on amino acids. In other embodiments, the linker isnot peptide-like. In some embodiments, the linker is carbon bond,disulfide bond, carbon-heteroatom bond, etc. In certain embodiments, thelinker is a carbon-nitrogen bond of an amide linkage. In certainembodiments, the linker is a cyclic or acyclic, substituted orunsubstituted, branched or unbranched aliphatic or heteroaliphaticlinker. In certain embodiments, the linker is polymeric (e.g.,polyethylene, polyethylene glycol, polyamide, polyester, etc.). Incertain embodiments, the linker comprises a monomer, dimer, or polymerof aminoalkanoic acid. In certain embodiments, the linker comprises anaminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine,3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). Incertain embodiments, the linker comprises a monomer, dimer, or polymerof aminohexanoic acid (Ahx). In certain embodiments, the linker is basedon a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In otherembodiments, the linker comprises a polyethylene glycol moiety (PEG). Inother embodiments, the linker comprises amino acids. In certainembodiments, the linker comprises a peptide. In certain embodiments, thelinker comprises an aryl or heteroaryl moiety. In certain embodiments,the linker is based on a phenyl ring. The linker may includefunctionalized moieties to facilitate attachment of a nucleophile (e.g.,thiol, amino) from the peptide to the linker. Any electrophile may beused as part of the linker. Exemplary electrophiles include, but are notlimited to, activated esters, activated amides, alkyl halides, arylhalides, acyl halides, and isothiocyanates.

In some embodiments, the linker is an amino acid or a plurality of aminoacids (e.g., a peptide or protein). In some embodiments, the linker is abond (e.g., a covalent bond), an organic molecule, group, polymer, orchemical moiety. In some embodiments, the linker is 5-100 amino acids inlength, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45,45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130,130-140, 140-150, or 150-200 amino acids in length. Longer or shorterlinkers are also contemplated. In some embodiments, a linker comprisesthe amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 17), which may alsobe referred to as the XTEN linker. In some embodiments, a linkercomprises the amino acid sequence SGGS. In some embodiments, a linkercomprises (SGGS)_(n) (SEQ ID NO: 41), (GGGS)_(n) (SEQ ID NO: 42),(GGGGS)_(n) (SEQ ID NO: 43), (G)_(n) (SEQ ID NO: 44), (EAAAK)_(n) (SEQID NO: 45), (GGS)_(n) (SEQ ID NO: 46), SGSETPGTSESATPES (SEQ ID NO: 17),or (XP)_(n) (SEQ ID NO: 47) motif, or a combination of any of these,wherein n is independently an integer between 1 and 30, and wherein X isany amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15. In some embodiments, a linker comprisesSGSETPGTSESATPES (SEQ ID NO: 17), SGGSSGSETPGTSESATPESSGGS (SEQ ID NO:19). In some embodiments, a linker comprisesSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 20). In some embodiments, alinker comprisesGGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 21). In someembodiments, the linker is 24 amino acids in length. In someembodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 23). In some embodiments, thelinker is 40 amino acids in length. In some embodiments, the linkercomprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 24). In someembodiments, the linker is 64 amino acids in length. In someembodiments, the linker comprises the amino acid sequenceSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQID NO: 25). In some embodiments, the linker is 92 amino acids in length.In some embodiments, the linker comprises the amino acid sequencePGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 26). It should beappreciated that any of the linkers provided herein may be used to linka first adenosine deaminase and a second adenosine deaminase; adeaminase (e.g., a first or a second adenosine deaminase) and a RNAguided programmable DNA binding protein; a RNA guided programmable DNAbinding protein and an NLS; or a deaminase (e.g., a first or a secondadenosine deaminase) and an NLS.

Various linker lengths and flexibilities between a deaminase (e.g., anengineered ecTadA) and a RNA guided programmable DNA binding protein(e.g., a Cas9 domain), and/or between a first adenosine deaminase and asecond adenosine deaminase can be employed (e.g., ranging from veryflexible linkers of the form (GGGGS)_(n) (SEQ ID NO: 22), (GGGGS)_(n)(SEQ ID NO: 22), and (G)_(n) to more rigid linkers of the form(EAAAK)_(n) (SEQ ID NO: 48), (SGGS)_(n) (SEQ ID NO: 49), and (XP)_(n))in order to achieve the optimal length for deaminase activity for thespecific application. In some embodiments, n is any integer between 3and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15. In some embodiments, the linker comprises a (GGS)_(n)(SEQ ID NO: 51) motif, wherein n is 1, 3, or 7.

Protospacer Adjacent Motifs

CRISPR clusters contain spacers, sequences complementary to antecedentmobile elements, and target invading nucleic acids. CRISPR clusters aretranscribed and processed into CRISPR RNA (crRNA). For example, in typeII CRISPR systems correct processing of pre-crRNA requires atrans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) anda Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aidedprocessing of pre-crRNA. Cas9 recognizes a short motif in the CRISPRrepeat sequences (the PAM or protospacer adjacent motif) (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes.”Ferretti et ah, J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G.,Lyon K., Primeaux C, Sezate S., Suvorov A. N., Kenton S., Lai H. S., LinS. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., Natl.Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation bytrans-encoded small RNA and host factor RNase III.” Deltcheva E.,Chylinski K., Sharma C M., Gonzales K., Chao Y., Pirzada Z. A., EckertM. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A.,Charpentier E. Science 337:816-821(2012), the entire contents of each ofwhich are incorporated herein by reference). Cas9 orthologs have beendescribed in various species, including, but not limited to, S. pyogenesand S. thermophilus.

Some aspects of the disclosure provide programmed DNA binding proteindomains, e.g. Cas9 domains that have altered PAM specificities. In someembodiments, a Cas9 derived from S. pyogenes (spCas9) recognizes acanonical NGG PAM sequence where the “N” in “NGG” is adenine (A),thymine (T), guanine (G), or cytosine (C), and the G is guanine. In someembodiments, the base editing fusion proteins provided herein functionby deaminating a target nucleobase within a 4 base region (e.g., a“deamination window”), which is approximately 15 bases upstream of thePAM. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 basesupstream of the PAM. Accordingly, in some embodiments, any of the fusionproteins provided herein may contain a Cas9 domain that is capable ofbinding a nucleotide sequence that does not contain a canonical (e.g.,NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequenceshave been described in the art and would be apparent to the skilledartisan. For example, Cas9 domains that bind non-canonical PAM sequenceshave been described in Kleinstiver, B. P., et al., “EngineeredCRISPR-Cas9 nucleases with altered PAM specificities” Nature 523,481-485 (2015); and Kleinstiver, B. P., et ah, “Broadening the targetingrange of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition”Nature Biotechnology 33, 1293-1298 (2015); the entire contents of eachare hereby incorporated by reference. In some embodiments, the Cas9domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In someembodiments, the SaCas9 domain is a nuclease active SaCas9, a nucleaseinactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). A wild typeSaCas9 amino acid sequence is provided below:

(SEQ ID NO: 52) KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK KG

In some embodiments, the SaCas9 comprises a N579X mutation, or acorresponding mutation in any of the amino acid sequences as numbered inthe wild type SaCas9 sequence, wherein X is any amino acid except for N.In some embodiments, the SaCas9 comprises a N579A mutation as numberedin the wild type SaCas9 sequence or a corresponding mutation in anotherSaCas9 protein.

In some embodiments, the SaCas9 domain, the nuclease inactive SaCas9domain, or the SaCas9 nickase domain can bind to a nucleic acid sequencehaving a non-canonical PAM. In some embodiments, the SaCas9 domain, theSaCas9d domain, or the SaCas9n domain can bind to a nucleic acidsequence having a NNGRRT PAM sequence, where N=A, T, C, or G, and R=A orG. In some embodiments, the SaCas9 domain comprises one or more ofE781X, N967X, and R1014X as numbered in the wild type SaCas9 sequence ora corresponding mutation in another SaCas9 protein wherein X is anyamino acid. In some embodiments, the SaCas9 domain comprises one or moreof a E781K, a N967K, and a R1014H mutation as numbered in the wild typeSaCas9 sequence or a corresponding mutation in another SaCas9 protein.In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or aR1014H mutation as numbered in the wild type SaCas9 sequence or acorresponding mutation in another SaCas9 protein.

In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcuspyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nucleaseactive SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase(SpCas9n). In some embodiments, the SpCas9 comprises a D10X mutation asnumbered in the wild type SpCas9 amino acid sequence or a correspondingmutation in another SapCas9 protein, wherein X is any amino acid exceptfor D. In some embodiments, the SpCas9 comprises a D10A mutation asnumbered in the wild type SpCas9 amino acid sequence or a correspondingmutation in another SpCas9 protein. In some embodiments, the SpCas9domain, the nuclease in active SpCas9 domain, or the SpCas9 nickasedomain can bind to a nucleic acid sequence having a non-NGG PAM. In someembodiments, the SpCas9 domain, the SpCas9 domain, the nuclease inactive SpCas9 domain, or the SpCas9 nickase domain can bind to a nucleicacid sequence having a NGG, a NGA, or a NGCG PAM sequence. In someembodiments, the SpCas9 domain comprises one or more of a D1135X, aR1335X, and a T1337X mutation as numbered in the wild type SpCas9 aminoacid sequence or a corresponding mutation in another SpCas9 protein,wherein X is any amino acid. In some embodiments, the SpCas9 domaincomprises one or more of a D1135E, R1335Q, and T1337R mutation asnumbered in the wild type SpCas9 amino acid sequence or a correspondingmutation in another SpCas9 protein. In some embodiments, the SpCas9domain comprises one or more of a D1 134V, a R1334Q, and a T1336Rmutation as numbered in the wild type Cas9 amino acid sequence, or acorresponding mutation thereof. In some embodiments, the SpCas9 domaincomprises a D1135V, a R1335Q, and a T1337R mutation as numbered in thewild type SpCas9 amino acid sequence or a corresponding mutation inanother SpCas9 protein. In some embodiments, the SpCas9 domain comprisesone or more of a D1135X, a G1218X, a R1335X, and a T1337X mutation asnumbered in the wild type SpCas9 amino acid sequence or a correspondingmutation in another SpCas9 protein, wherein X is any amino acid. In someembodiments, the SpCas9 domain comprises one or more of a D1135V, aG1218R, a R1335Q, and a T1337R mutation as numbered in the wild typeSpCas9 amino acid sequence or a corresponding mutation in another SpCas9protein. In some embodiments, the SpCas9 domain comprises a D1135V, aG1218R, a R1335Q, and a T1337R mutation as numbered in the wild typeSpCas9 amino acid sequence or a corresponding mutation in another SpCas9protein.

In some embodiments, the Cas9 domain of any of the fusion proteinsprovided herein comprises an amino acid sequence that is at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5% identical to any one of the Cas9 sequencesprovided herein.

In some embodiments, a base editor provided herein comprises a RNA guideprogrammable DNA binding protein that may be used to target and modify aspecific nucleic acid (e.g., DNA or RNA) sequence. Nucleic acidprogrammable DNA binding proteins include, without limitation, Cas9(e.g., nuclease inactive Cas9 and Cas9 nickase), CasX, CasY, Cas12a(Cpf1), Cas12b, C2c1, C2c2, C2C3, and Argonaute. One example of annucleic acid programmable DNA-binding protein that has different PAMspecificity than Cas9 is Clustered Regularly Interspaced ShortPalindromic Repeats from Prevotella and Francisella 1 (Cpf1). Similar toCas9, Cpf1 is also a class 2 CRISPR effector. It has been shown thatCpf1 mediates robust DNA interference with features distinct from Cas9.Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and itutilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Cpf1proteins described in Yamano et ah, “Crystal structure of Cpf1 incomplex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; theentire contents of which is hereby incorporated by reference.

In some embodiments, a base editor provided herein comprises a nucleaseinactive Cpf1 protein or a variant thereof. The Cpf1 protein has aRuvC-like endonuclease domain that is similar to the RuvC domain of Cas9but does not have a HNH endonuclease domain, and the N-terminal of Cpf1does not have the alfa-helical recognition lobe of Cas9. In someembodiments, the Cpf1 nickase comprises one or more mutationscorresponding to D917A, E1006A, or D1255A as numbered in the Francisellanovicida Cpf1 protein.

Wild type Francisella novicida Cpf1 amino acid sequence is providedbelow:

(SEQ ID NO: 53) MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

In some embodiments, the base editor comprises a Cpf1 protein. In someembodiments, the Cpf1 protein is a Cpf1 nickase. In some embodiments,the Cpf1 protein is a nuclease inactive Cpf1. In some embodiments, theCpf1 protein comprises an amino acid sequence that is at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atease 99.5% identical to the FnCpf1 sequence provided herein. In someembodiments, the Cpf1 protein comprises mutations corresponding toD917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, orD917A/E1006A/D1255A as numbered in the wild type FnCpf1 sequenceprovided herein. It should be appreciated that Cpf1 from other bacterialspecies may also be used in accordance with the present disclosure.

In some embodiments, the nucleic acid programmable DNA binding proteincomprises a Cas12b (C2c1), C2c2, or C2c3 protein or a variant thereof.Additional features of Cas proteins in Class 2 CRISPR-Cas systemsdescribed in Shmakov et al., “Discovery and Functional Characterizationof Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3):385-397, the entire contents of which is hereby incorporated byreference. Effectors of two of the systems, C2c1 and C2c3, containRuvC-like endonuclease domains related to Cpf1. A third system, C2c2contains an effector with two predicated HEPN RNase domains. Productionof mature CRISPR RNA is tracrRNA-independent, unlike production ofCRISPR RNA by C2c1. C2c1 depends on both CRISPR RNA and tracrRNA for DNAcleavage. The crystal structure of Alicyclobaccillus acidoterrastrisC2c1 (AacC2c1) in complex with a chimeric single-molecule guide RNA(sgRNA) is described in Liu et al., “C2c1-sgRNA Complex StructureReveals RNA→Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan.19;65(2):310-322; Yang et al., “PAM-dependent Target DNA Recognition andCleavage by C2C1 CRISPR-Cas, the entire contents of which are herebyincorporated by reference.

An exemplary Cas12b amino acid sequence (Bacillus hisashii Cas12b) isprovided below:

BhCasl2b (Bacillus hisashii) NCBI Reference Sequence: WP_095142515(SEQ ID NO: 54) MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMK RPAATKKAGQAKKKK

In some embodiments, the programmable DNA binding protein comprises anargonaute protein. One example of such a nucleic acid programmable DNAbinding protein is an Argonaute protein from Natronobacterium gregoryi(NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5′phosphorylated ssDNA of −24 nucleotides (gDNA) to guide it to its targetsite and will make DNA double-strand breaks at the gDNA site. Incontrast to Cas9, the NgAgo-gDNA system does not require aprotospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo(dNgAgo) can greatly expand the bases that may be targeted. Thecharacterization and use of NgAgo have been described in Gao et al., NatBiotechnol., 2016 July; 34(7):768-73. PubMed PMID: 27136078; Swarts etal., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic AcidsRes. 43(10) (2015):5120-9, each of which is incorporated herein byreference.

In some embodiments, the protospacer sequence comprises a nucleotidesequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the protospacer sequenceslisted in Table 1 or Table 24. In some embodiments, the protospacersequence comprises any one of the protospacer sequences listed in Table1 or Table 24. In some embodiments, the protospacer sequence is any oneof the protospacer sequences listed in Table 1 or Table 24. In someembodiments, the protospacer sequence comprises a nucleotide sequencethat is at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or atleast 99.9% identical to any one of the sequences of SEQ ID NOs: 13-15,50, 66-69, 81-252, and 1594-1617. In some embodiments, the protospacersequence comprises any one of the sequences of SEQ ID NOs: 13-15, 50,66-69, 81-252, and 1594-1617. In some embodiments, the protospacersequence is any one of the sequences of SEQ ID NOs: 13-15, 50, 66-69,81-252, and 1594-1617.

Guide Polynucleotides

Some aspects of this disclosure provide complexes comprising any of thefusion proteins provided herein, and a guide polynucleotide bound to aprogrammable DNA binding protein, e.g. a Cas9 domain of the base editoror fusion protein.

In some embodiments, a guide polynucleotide is a guide polynucleotide, aguide RNA (gRNA), or a nucleic acid encoding the same.

In some embodiments, the guide polynucleotide comprises a single nucleicacid sequence. In some embodiments, the guide polynucleotide comprisestwo nucleic acid sequences. In some embodiments, the guide nucleic acid(e.g., guide RNA) is from 15-100 nucleotides long and comprises asequence of at least 10 contiguous nucleotides that is complementary toa target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotideslong. In some embodiments, the guide RNA comprises a sequence of 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementaryto a target sequence. In some embodiments, the target sequence is a DNAsequence. In some embodiments, the target sequence is a sequence in thegenome of a mammal. In some embodiments, the target sequence is asequence in the genome of a human. In some embodiments, the 3′ end ofthe target sequence is immediately adjacent to a canonical PAM sequence(NGG). In some embodiments, the guide nucleic acid (e.g., guide RNA) iscomplementary to a sequence associated with a disease or disorder. Insome embodiments, the guide nucleic acid (e.g., guide RNA) iscomplementary to a sequence associated with a disease or disorder havinga mutation in a PCSK9 gene, a ANGPTL3 gene, or a APOC3 gene.

Some aspects of this disclosure provide methods of using the fusionproteins, or complexes comprising a guide nucleic acid (e.g., gRNA) anda nucleobase editor provided herein. For example, some aspects of thisdisclosure provide methods comprising contacting a DNA, or RNA moleculewith any of the fusion proteins provided herein, and with at least oneguide nucleic acid (e.g., guide RNA), wherein the guide nucleic acid,(e.g., guide RNA) is about 15-100 nucleotides long and comprises asequence of at least 10 contiguous nucleotides that is complementary toa target sequence. In some embodiments, the 3′ end of the targetsequence is immediately adjacent to a canonical PAM sequence (NGG). Insome embodiments, the 3′ end of the target sequence is not immediatelyadjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′end of the target sequence is immediately adjacent to an AGC, GAG, TTT,GTG, or CAA sequence.

In some embodiments, a guide polynucleotide is a DNA. In someembodiments, a guide polynucleotide is an RNA. In some embodiments, aguide polynucleotide is a modified, artificial polynucleotides. In someembodiments, a guide polynucleotide is a single or single moleculepolynucleotide. In some embodiments, a guide polynucleotide comprisesdual polynucleotides. In some embodiments, a guide polynucleotide is adual polynucleotides connected by a linker. In some embodiments, a guidepolynucleotide is a dual polynucleotides connected by a non-nucleic acidlinker. In some embodiments, a guide polynucleotide is a dualpolynucleotides connected by a peptide linker or a chemical linker. Insome embodiments, the guide polynucleotide is a single guide RNA. Theguide RNA (gRNA) may guide the programmable DNA binding protein, e.g., aClass 2 Cas nuclease, e.g. a Cas9 to a target sequence on a targetnucleic acid molecule, where the gRNA hybridizes with and theprogrammable DNA binding protein and modifies the target sequence. Insome embodiments, the gRNA and the base editor fusion protein may form aribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. In someembodiments, the CRISPR complex may be a Type II CRISPR/Cas9 complex. Insome embodiments, the CRISPR/Cas complex may be a Type V CRISPR/Cascomplex, such as a Cpf1/guide RNA complex.

A gRNA can comprise at least three regions: a first region at the 5′ endthat can bind to the complementary strand of a target site in achromosomal sequence (spacer region), a second internal region that canform a stem loop structure, and a third 3′ region that can besingle-stranded. A first region of each guide RNA can also be differentsuch that each gRNA guides a protein, e.g., Cas9, to a specific targetsite. Further, second and third regions of each gRNA can be identical inall gRNAs. A second region of a gRNA may form a secondary structure. Insome embodiments, a secondary structure formed by a gRNA can comprise astem (or hairpin) and a loop. A length of a loop and a stem can vary. Insome embodiments, a loop can range from about 3 to about 10 nucleotidesin length. In some embodiments, a stem can range from about 6 to about20 nucleotides in length. A stem can comprise one or more bulges of 1 to10 nucleotides or about 10 nucleotides. In some embodiments, the overalllength of a second region can range from about 16 to 60 nucleotides inlength. In some embodiments, a loop can be about 4 nucleotides inlength. In some embodiments, a stem can be about 12 in length. A thirdregion of gRNA at the 3′ end can be essentially single-stranded. In someembodiments, a third region is sometimes not complementary to anychromosomal sequence in a cell of interest and is sometimes notcomplementary to the rest of a gRNA. In addition, the length of a thirdregion can vary. In some embodiments, a third region can be more than 3or more than 4 nucleotides in length. For example, the length of a thirdregion can range from about 5 to 60 nucleotides in length.

A gRNA for a base editor system can comprise a CRISPR RNA (crRNA) and atrans-activating crRNA (tracrRNA). In some embodiments, the crRNA maycomprise a targeting sequence (or spacer sequence) that hybridizes withthe complementary strand of the target sequence on the target nucleicacid molecule. The crRNA may also comprise a flagpole that iscomplementary to and hybridizes with a portion of the tracrRNA. In someembodiments, the crRNA may parallel the structure of a naturallyoccurring crRNA transcribed from a CRISPR locus of a bacteria, where thetargeting sequence acts as the protospacer of the Cas9 in the baseeditor system. The gRNA may target any sequence of interest via thetargeting sequence of the crRNA. In some embodiments, the degree ofcomplementarity between the targeting sequence of the gRNA and thetarget sequence on the target nucleic acid molecule is at least about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In someembodiments, the targeting sequence of the gRNA and the target sequenceon the target nucleic acid molecule may be 100% complementary. In otherembodiments, the targeting sequence of the gRNA and the target sequenceon the target nucleic acid molecule may contain at least one mismatch.For example, the targeting sequence of the gRNA and the target sequenceon the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 mismatches.

In some embodiments, the length of the targeting sequence depends on theCRISPR/Cas component of the base editor system and components used. Forexample, different Cas proteins from different bacterial species havevarying optimal targeting sequence lengths. Accordingly, the targetingsequence may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50nucleotides in length. In some embodiments, the targeting sequencecomprised 18-24 nucleotides in length. In some embodiments, thetargeting sequence comprises 19-21 nucleotides in length. In someembodiments, the targeting sequence comprises 20 nucleotides in length.

In some embodiments, the guide RNA is a “dual guide RNA” or “dgRNA”. Insome embodiments, the dgRNA comprises a first RNA molecule comprising acrRNA, and a second RNA molecule comprising a tracrRNA. The first andsecond RNA molecules may form a RNA duplex via the base pairing betweenthe crRNA and the tracrRNA (e.g. the repeat and anti repeat). In someembodiments, the guide RNA is a “single guide RNA” or “sgRNA.” In someembodiments, the sgRNA may comprise a crRNA covalently linked to atracrRNA. In some embodiments, the crRNA and the tracrRNA may becovalently linked via a linker. In some embodiments, the single-moleculeguide RNA may comprise a stem-loop structure via the base pairingbetween the crRNA and the tracrRNA. In some embodiments, the sgRNA is a“Cas9 sgRNA” capable of directing a Cas9 protein. In certainembodiments, the guide RNA comprises a crRNA and tracrRNA sufficient forforming an active complex with a Cas9 protein and mediating RNA-guidedDNA modification. The term “gRNA” and “sgRNA” are used interchangeablythroughout this application. In some embodiments, more than one guideRNAs can be used; each guide RNA contains a different targetingsequence, such that the base editor system modifies more than one targetsequence. In some embodiments, one or more guide RNAs may have the sameor differing properties such as activity or stability within a baseeditor complex. Where more than one guide RNA is used, each guide RNAcan be encoded on the same or on different expression cassettes. Thepromoters used to drive expression of the more than one guide RNA may bethe same or different.

In some embodiments, the gRNA or mRNA encoding Cas protein is modified.The modifications can comprise chemical alterations, syntheticmodifications, nucleotide additions, and/or nucleotide subtractions andmodified nucleosides or nucleotides can be present in a gRNA. A gRNA orCas protein-encoding mRNA comprising one or more modified nucleosides ornucleotides is called a “modified” RNA to describe the presence of oneor more non-naturally and/or naturally occurring components orconfigurations that are used instead of or in addition to the canonicalA, G, C, and U residues. In some embodiments, a modified RNA issynthesized with a non-canonical nucleoside or nucleotide. Modifiednucleosides and nucleotides can include one or more of: (i) alteration,e.g., replacement, of one or both of the non-linking phosphate oxygensand/or of one or more of the linking phosphate oxygens in thephosphodiester backbone linkage (an exemplary backbone modification);(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugarmodification); (iii) wholesale replacement of the phosphate moiety with“dephospho” linkers (an exemplary backbone modification); (iv)modification or replacement of a naturally occurring nucleobase,including with a non-canonical nucleobase (an exemplary basemodification); (v) replacement or modification of the ribose-phosphatebackbone (an exemplary backbone modification); (vi) modification of the3′ end or 5′ end of the oligonucleotide, e.g., removal, addition,modification, or replacement of a terminal phosphate group orconjugation of a moiety, cap, or linker (such 3′ or 5′ cap modificationsmay comprise a sugar and/or backbone modification); and (vii)modification or replacement of the sugar (an exemplary sugarmodification). The modifications can enhance genome editing byCRISPR/Cas. A modification can alter chirality of a gRNA. In some cases,chirality may be uniform or stereopure after a modification. A guide RNAcan also be truncated. Truncation can be used to reduce undesiredoff-target mutagenesis. The truncation can comprise any number ofnucleotide deletions. For example, the truncation can comprise 1, 2, 3,4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides. In someembodiments, the term “cap,” as used herein, may be used to refer to oneor more specially altered or modified nucleotides, e.g., speciallyaltered or modified nucleotides on the 5′ end of mRNA. In someembodiments, the cap comprises to a modified guanine (G) nucleotide. Insome embodiments, the cap refers to the 5′ end modification of mRNAs byaddition of 7-Methylguanosine (N7-methyl guanosine or m7G). In someembodiments, “capping” of the mRNA structure plays a crucial role in avariety of cellular processes which include translation initiation,splicing, intracellular transport, and turnover. In some embodiment, the5′ cap enhances mRNA stability as well as translation efficiency.Exemplary cap analogs include, but are not limited to, standard capanalog m7G(5′)ppp(5′)G, anti-reverse cap analog (ARCA) m7,3′-OGpppG (or3′-O-Me-m7G(5′) ppp(5′)G), unmethylated cap analog G (5′)ppp(5′)G,methylated cap analog for A+1 sites m7G(5′)ppp(5′)A, and unmethylatedcap analog for A+1 sites G(5′)ppp(5′)A. Additional methods of obtainingmRNA cap analogs are described in Kowalska et al., RNA 2008 14(6):1119-1131, which is incorporated herein in its entirety.

In some embodiments, the guide RNA sequence comprises a nucleotidesequence that is at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%,or at least 99.9% identical to any one of the guide sequences listed inTable 1 or Table 24. In some embodiments, the guide RNA sequencecomprises any one of the guide RNA sequences listed in Table 1 or Table24. In some embodiments, the guide RNA sequence is any one of the guideRNA sequences listed in Table 1 or Table 24. In some embodiments, theguide RNA sequence comprises a nucleotide sequence that is at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, at least 99.7%, or at least 99.9% identicalto any one of the sequences of SEQ ID NOs: 9-11, 55, 59, 253-452,1618-1635, 1637-1800, 1802-2135, and 2191. In some embodiments, theguide RNA sequence comprises any one of the sequences of SEQ ID NOs:9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. Insome embodiments, the guide RNA sequence is any one of the sequences ofSEQ ID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and2191.

Off-Target Effects

With standard CRISPR-Cas9 genome editing, off-target mutagenesis—i.e.,edits at genomic sites other than the intended target site—can occur atsites that share a high degree of sequence similarity with theprotospacer sequence encoded in the gRNA.

As used herein, off-target effect may be used refer to modifications,including indels, at genomic sites other than intended target site, forexample, a protospacer sequence recognized by the guide RNA sequence. Inthe context of base editing, off-target editing may occur at genomicsites remote from an intended target site, or in close proximity to theintended target site. For example, one or more bases besides the targetnucleobase in the base editing window may be edited (“bystanderediting”). In certain embodiments, off-target base editing, e.g.bystander editing does not impact expression or function of the targetgene.

Potential off-target sequence candidates in the genome may be predictedby bioinformatic analyses of the human genome using, e.g., the MITSpecificity Score (calculated by http://crispor.tefor.net/; minimumscore of 50) or by in vitro biochemical assays, e.g., ONE-seq.

Off-target editing may be determined by sequencing analysis. In someembodiments, off-target editing is calculated using net nucleobaseediting at potential off-target sites in the genome. For example, netnucleobase editing efficiency may be determined using no-base editor orno-guide RNA as a control, where net nucleobase editing efficiency isobtained by editing rate observed in cells contacted with a LNPenclosing a base editor mRNA and a guide RNA subjected by editing rateobserved in control cells at predicted off-target sites. Additionalmethods for estimating and reducing off-target editing are described inRees et al., Nat. Rev. Genet. 2018 19(12): 770-788, which isincorporated herein by reference in its entirety.

Methods for selecting, designing, and validating gRNAs and targetingsequences (or spacer sequences) are described herein and known to thoseskilled in the art. Software tools can be used to optimize the gRNAscorresponding to a target nucleic acid sequence, e.g., to minimize totaloff-target activity across the genome. For example, for each possibletargeting domain choice using S. pyogenes Cas9, all off-target sequences(preceding selected PAMs, e.g., NAG or NGG) may be identified across thegenome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10) of mismatched base-pairs. First regions of gRNAs complementaryto a target site can be identified, and all first regions (e.g., crRNAs)can be ranked according to its total predicted off-target score; thetop-ranked targeting domains represent those that are likely to have thegreatest on-target and the least off-target activity. In someembodiments, a DNA sequence searching algorithm can be used to identifya target sequence in crRNAs of a gRNA for use with Cas9. A custom gRNAdesign software based on the public tool cas-offinder, which scoresguides after calculating their genome-wide off-target propensity, can bealso used to design a gRNA (Bae, et al., Cas-OFFinder: A fast andversatile algorithm that searches for potential off-target sites of Cas9RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014)). In someembodiments, RepeatMasker program can be used to screen repeat elementsand regions of low complexity in the input DNA sequences. In addition,the number of residues that could unintentionally be targeted (e.g.,off-target residues that could potentially reside on ssDNA within thetarget nucleic acid locus) may be minimized to reduce the impact ofpotential substrate promiscuity of a deaminase domain in the nucleobaseeditor system. Candidate gRNAs can be functionally evaluated by usingmethods known in the art and/or as set forth herein.

In some embodiments, target sequences for a Cas9 protein in a baseeditor system can be selected based on the presence of target sequencesof a gene of interest in one or more species. For example, a sequencemay be selected as a target sequence if the sequence match targetsequences in both human and cynomolgus monkey orthologs of a gene ofinterest. In some embodiments, target sequences for a Cas9 nuclease canbe selected based on predicted off-target profiles, as judged by the MITSpecificity Score (calculated by http://crispor.tefor.net/). Forexample, a sequence may be selected as a target sequence if the sequencehas a favorable predicted off-target profiles (e.g., minimum score of 50when judged by the MIT Specificity Score). In some embodiments, targetsequences for an adenine base editor (ABE) can be selected based on theability of editing an adenine base within a splice donor site or asplice acceptor site in a target gene. In some embodiments, targetsequences for an ABE can be selected based on the position of anadenine, e.g., if the adenine lies within the editing window of the ABE.

The gRNAs described herein can be synthesized chemically, enzymatically,or a combination thereof. For example, the gRNA can be synthesized usingstandard phosphoramidite-based solid-phase synthesis methods.Alternatively, the gRNA can be synthesized in vitro by operably linkingDNA encoding the gRNA to a promoter control sequence that is recognizedby a phage RNA polymerase. Examples of suitable phage promoter sequencesinclude, but are not limited to, T7, T3, SP6 promoter sequences, orvariations thereof. In some embodiments, gRNA comprises two separatemolecules (e.g., crRNA and tracrRNA) and one molecule (e.g., crRNA) canbe chemically synthesized and the other molecule (e.g., tracrRNA) can beenzymatically synthesized.

In some aspects, provided herein, is a composition for gene modificationcomprising a single guide RNA (sgRNA) described herein and the baseeditor fusion protein or a nucleic acid sequence encoding the baseeditor fusion protein. In some embodiments, the composition furthercomprises a vector that comprises the nucleic acid sequence encoding thebase editor fusion protein. In some embodiments, base editor fusionprotein comprises a Cas9 protein. In some embodiments, the Cas9 proteinis a the Cas9 protein is a Streptococcus pyogenes Cas9. In someembodiments, the composition further comprises a pharmaceuticallyacceptable carrier. Non-limiting examples of pharmaceutically acceptablecarriers are listed in “Pharmaceutical composition and methods oftreatment” section of this application. In some embodiments, thecomposition described herein can be provided in a lipid nanoparticle.

Chemically Modified Guide RNAs (2RNAs)

The present disclosure, provided herein, relates to a chemicallymodified CRISPR guide RNA (gRNA) that may be used in association withany general CRISPR/Cas system, for example, a base editor system asdescribed herein. gRNAs (e.g., sgRNAs, short-sgRNAs, crRNAs, tracrRNAs,or dgRNAs) may comprise modifications at various nucleotide positions.In some aspects, provided herein, is a guide RNA (gRNA) or a singleguide RNA (sgRNA) that comprises (i) a chemical modification at anucleotide and (ii) an unmodified nucleotide. In some embodiments, thegRNA or the sgRNA may comprise a modification at a specific nucleotideposition. In some embodiments, the gRNA or the sgRNA may comprise one ormore modifications at one or more specific positions.

In an aspect, chemically modified CRISPR guide RNAs provided hereincomprise modifications based on crystal structures. Not intended to bebound by a particular theory, X-ray crystal structure based guide designmay be used to optimize gRNA modification in complex with the Casprotein, e.g. Cas9. In some embodiments, a nucleotide where a 2′-OH isin close proximity with a Cas9 protein in the Cas9-gRNA complex isunmodified. In some embodiments, a nucleotide where a 2′-OH is incontact with a Cas9 protein in the Cas9-gRNA complex is unmodified. Insome embodiments, a nucleotide where a 2′-OH is in contact with a Cas9protein with a hydrogen bond in the Cas9-gRNA complex is unmodified. Insome embodiments, the Cas9-gRNA complex is a pre-catalytic ternarycomplex. In some embodiments, a nucleotide where a 2′-OH is in closeproximity with another part of the gRNA in the Cas9-gRNA complex isunmodified. In some embodiments, a nucleotide where a 2′-OH is incontact with another part of the gRNA in the Cas9-gRNA complex isunmodified. In some embodiments, a nucleotide where a 2′-OH is incontact with another part of the gRNA with a hydrogen bond in theCas9-gRNA complex is unmodified. In some embodiments, the Cas9-gRNAcomplex is a pre-catalytic ternary complex. As a person of skill in theart would understand, the term “clash” as used herein refers tophysically-unlikely overlapping atomic volumes in a structure.Incorporation of a 2′O-Me modification in these situations wouldpotentially result in structural rearrangement(s) that could bedetrimental to RNP function. As a person of skill in the art wouldunderstand, the term “contact” as used herein means a stable,non-covalent interaction between two functional groups, e.g. a hydrogenbond. In some embodiments, a nucleotide where a clash with another partof the gRNA is predicted if a 2′-OH is replaced with a 2′-OMe in theCas9-gRNA complex is unmodified. In some embodiments, a nucleotide wherea clash with another 2′-OH in the gRNA is predicted if a 2′-OH isreplaced with a 2′-OMe in the Cas9-gRNA complex is unmodified. In someembodiments, the Cas9-gRNA complex is a pre-catalytic ternary complex.In some embodiments, a nucleotide that is solvent-exposed or otherwisedistant from Cas protein residues in the Cas9-gRNA complex comprises achemical modification. In some embodiments, a nucleotide that issolvent-exposed or otherwise distant from another nucleotide in the gRNAin the Cas9-gRNA complex comprises a chemical modification. In someembodiments, the chemical modification is a 2′-OMe modification.Additional description of Cas9-gRNA crystal structure is included inJiang F, Taylor D W, Chen J S, Kornfeld J E, Zhou K, Thompson A J,Nogales E, Doudna J A. Structures of a CRISPR-Cas9 R-loop complex primedfor DNA cleavage. Science. 2016 Feb. 19;351(6275):867-71, incorporatedherein by reference in its entirety.

Modifications to guide RNA nucleotides can include, but are not limitedto, 2′-O-methyl modifications, 2′-O-(2-methoxyethyl) modifications,2′-fluoro modifications, phosphorothioate modifications, inverted abasicmodifications, deoxyribonucleotides, bicylic ribose analog (e.g., lockednucleic acid (LNA), C-ethylene-bridged nucleic acid (ENA), bridgednucleic acid (BNA), unlocked nucleic acid (UNA)), base or nucleobasemodifications, internucleoside linkage modifications, ribonebularine,2′-O-methylnebularine, or 2′-deoxynebularine. Other examples ofmodifications include, but are not limited to, 5′adenylate, 5′guanosine-triphosphate cap, 5′N7-Methylguanosine-triphosphate cap,5′triphosphate cap, 3′phosphate, 3′thiophosphate, 5′phosphate,5′thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer9,3′-3′ modifications, 5′-5′ modifications, abasic, acridine,azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG,desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PCbiotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1,black hole quencer 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35,QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′deoxyribonucleosideanalog purine, 2′deoxyribonucleoside analog pyrimidine, ribonucleosideanalog, 2′-O-methyl ribonucleoside analog, sugar modified analogs,wobble/universal bases, fluorescent dye label, 2′fluoro RNA, 2′O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA,phosphothioate DNA, phosphorothioate RNA, UNA,pseudouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate,2-O-methyl 3phosphorothioate or any combinations thereof.

In some embodiments, the ribose group (or sugar) may be modified. Insome embodiments, modified ribose group may control oligonucleotidebinding affinity for complementary strands, duplex formation, orinteraction with nucleases. Examples of chemical modifications to theribose group include, but are not limited to, 2′-O-methyl (2′-OMe),2′-fluoro (2′-F), 2′-deoxy, 2′-O-(2-methoxyethyl) (2′-MOE), 2′-NH2,2′-O-Allyl, 2′-O-Ethylamine, 2′-O-Cyanoethyl, 2′-O-Acetalester, or abicyclic nucleotide such as locked nucleic acid (LNA), 2′-(5-constrainedethyl (S-cEt)), constrained MOE, or 2′-0,4′-C-aminomethylene bridgednucleic acid (2′,4′-BNANC). In some embodiments, 2′-O-methylmodification can increase binding affinity of oligonucleotides. In someembodiments, 2′-O-methyl modification can enhance nuclease stability ofoligonucleotides. In some embodiments, 2′-fluoro modification canincrease oligonucleotide binding affinity and nuclease stability. Inthis application, a ribonucleotide in lowercase in gRNA sequence depicts2′-OMe modification (e.g., Table 1 or Table 24) (lowercase letter sindicates a phosphorothioate linkage).

In some embodiments, the phosphate group may be chemically modified.Examples of chemical modifications to the phosphate group includes, butare not limited to, a phosphorothioate (PS), phosphonoacetate (PACE),thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, orphosphotriester modification. In some embodiments, PS linkage can referto a bond where a sulfur is substituted for one nonbridging phosphateoxygen in a phosphodiester linkage, e.g., between nucleotides. An “s”may be used to depict a PS modification in gRNA sequences in thisapplication (e.g., Table 1 or Table 24). In some embodiments, a gRNA oran sgRNA may comprise a phosphorothioate (PS) linkage at a 5′ end or ata 3′ end. In some embodiments, a gRNA or an sgRNA may comprise aphosphorothioate (PS) linkage at a 5′ end. In some embodiments, a gRNAor an sgRNA may comprise a phosphorothioate (PS) linkage at a 3′ end. Insome embodiments, a gRNA or an sgRNA may comprise a phosphorothioate(PS) linkage at a 5′ end and at a 3′ end. In some embodiments, a gRNA oran sgRNA may comprise one, two, or three, or more than threephosphorothioate linkages at the 5′ end or at the 3′ end. In someembodiments, a gRNA or an sgRNA may comprise three phosphorothioate (PS)linkages at the 5′ end or at the 3′ end. In some embodiments, a gRNA oran sgRNA may comprise three phosphorothioate linkages at the 3′ end. Insome embodiments, a gRNA or an sgRNA may comprise two and no more thantwo (i.e., only two) contiguous phosphorothioate (PS) linkages at the 5′end or at the 3′ end. In some embodiments, a gRNA or an sgRNA maycomprise three contiguous phosphorothioate (PS) linkages at the 5′ endor at the 3′ end. In some embodiments, a gRNA or an sgRNA may comprisethe sequence 5′-UsUsU-3′ at the 3′end or at the 5′ end, wherein Uindicates a uridine and wherein s indicates a phosphorothioate (PS)linkage. In some embodiments, a gRNA or an sgRNA may comprise thesequence 5′-ususu-3′ at the 3′end or at the 5′ end, wherein u indicatesa 2′-O-methyluridine and wherein s indicates a phosphorothioate (PS)linkage. In some embodiments, a gRNA or an sgRNA may comprise thesequence 5′-ususuUUU-3′ at the 3′end or at the 5′ end, wherein U and uindicate uridine and 2′-O-methyluridine respectively, and wherein sindicates a phosphorothioate (PS) linkage. In some embodiments, a gRNAor an sgRNA may comprise the sequence 5′-ususuUuU-3′ at the 3′end or atthe 5′ end, wherein U and u indicate uridine and 2′-O-methyluridinerespectively, and wherein s indicates a phosphorothioate (PS) linkage.In some embodiments, a gRNA or an sgRNA may comprise the sequence5′-usususuUuU-3′ at the 3′end, wherein U and u indicate uridine and2′-O-methyluridine respectively, and wherein s indicates aphosphorothioate (PS) linkage. In some embodiments, a gRNA or an sgRNAmay comprise the sequence 5′-ususuuuu-3′ at the 3′end, wherein uindicates 2′-O-methyluridine, and wherein s indicates a phosphorothioate(PS) linkage.

In some embodiments, the nucleobase may be chemically modified. Examplesof chemical modifications to the nucleobase include, but are not limitedto, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine,2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substitutedpyrimidine, isoguanine, isocytosine, or halogenated aromatic groups.

In some embodiments the chemically modified gRNAs comprise nebularine.Nebularine is a purine ribonucleoside that is derived from abeta-D-ribose and is 9H-purine attached to a beta-D-ribofuranosylresidue at position 9 via a glycosidic (N-glycosyl) linkage. Nebularineis a purine ribonucleoside with no exocyclic functional moiety orsubstitution. In some embodiments, it is a purine ribonucleoside. Insome embodiments, it is a purine D-ribonucleoside. In some embodiments,nebularine is further modified chemically. In this application, “X,”“x,” and “dX” may be used to depict a ribonebularine modification, a2′-O-methylnebularine modification, and a 2′-deoxynebularinemodification, respectively, in gRNA sequences (e.g., Table 1 or Table24). In some embodiments, substitution of a nucleotide (e.g., A) of agRNA sequence (e.g., spacer region or tracr region) with nebularine mayreduce off-target effects without affecting the gRNA activity. In someembodiments, the nebularine, the deoxynebularine, or2′-O-methylnebularine replaces an adenine in an unmodified gRNA orsgRNA. In some embodiments, the nebularine, the deoxynebularine, or2′-O-methylnebularine is in the spacer sequence. In some embodiments,the nebularine, the deoxynebularine, or 2′-O-methylnebularine is in thetracrRNA sequence. In some embodiments, the nebularine, thedeoxynebularine, or 2′-O-methylnebularine is in a tracrRNA sequence inthe tracrRNA sequence. In some embodiments, the nebularine, thedeoxynebularine, or 2′-O-methylnebularine is in a crRNA sequence in thetracrRNA sequence. In some embodiments, the nebularine, thedeoxynebularine, or 2′-O-methylnebularine is in a stem loop structure inthe tracrRNA sequence.

In some embodiments, the chemically modified gRNAs may comprise a totalof 50-150 base pairs in length. In some embodiments, the chemicallymodified gRNAs may comprise a total of 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150base pairs in length. In some embodiments, the chemically modified gRNAsmay comprise a total of about 50 to about 140 base pairs in length. Insome embodiments, the chemically modified gRNAs may comprise a total ofabout 50 to about 60, about 50 to about 70, about 50 to about 80, about50 to about 90, about 50 to about 100, about 50 to about 110, about 50to about 120, about 50 to about 130, about 50 to about 140, about 60 toabout 70, about 60 to about 80, about 60 to about 90, about 60 to about100, about 60 to about 110, about 60 to about 120, about 60 to about130, about 60 to about 140, about 70 to about 80, about 70 to about 90,about 70 to about 100, about 70 to about 110, about 70 to about 120,about 70 to about 130, about 70 to about 140, about 80 to about 90,about 80 to about 100, about 80 to about 110, about 80 to about 120,about 80 to about 130, about 80 to about 140, about 90 to about 100,about 90 to about 110, about 90 to about 120, about 90 to about 130,about 90 to about 140, about 100 to about 110, about 100 to about 120,about 100 to about 130, about 100 to about 140, about 110 to about 120,about 110 to about 130, about 110 to about 140, about 120 to about 130,about 120 to about 140, or about 130 to about 140 base pairs in length.In some embodiments, the chemically modified gRNAs may comprise a totalof about 50, about 60, about 70, about 80, about 90, about 100, about110, about 120, about 130, or about 140 base pairs in length. In someembodiments, the chemically modified gRNAs may comprise a total of atleast about 50, about 60, about 70, about 80, about 90, about 100, about110, about 120, or about 130 base pairs in length. In some embodiments,the chemically modified gRNAs may comprise a total of at most about 60,about 70, about 80, about 90, about 100, about 110, about 120, about130, or about 140 base pairs in length. In one embodiment, thechemically modified gRNAs may comprise a total of 100 base pairs inlength. In another embodiment, the chemically modified gRNAs maycomprise a total of 103 base pairs in length.

In some embodiments, the chemically modified gRNAs may comprise from 1to 150 chemically modified nucleotides. In some embodiments, thechemically modified gRNAs may comprise a total of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 chemically modifiednucleotides. In some embodiments, the chemically modified gRNAs maycomprise a total of about 1 to about 45 chemically modified nucleotides.In some embodiments, the chemically modified gRNAs may comprise a totalof about 1 to about 3, about 1 to about 5, about 1 to about 10, about 1to about 15, about 1 to about 20, about 1 to about 25, about 1 to about30, about 1 to about 35, about 1 to about 40, about 1 to about 45, about3 to about 5, about 3 to about 10, about 3 to about 15, about 3 to about20, about 3 to about 25, about 3 to about 30, about 3 to about 35, about3 to about 40, about 3 to about 45, about 5 to about 10, about 5 toabout 15, about 5 to about 20, about 5 to about 25, about 5 to about 30,about 5 to about 35, about 5 to about 40, about 5 to about 45, about 10to about 15, about 10 to about 20, about 10 to about 25, about 10 toabout 30, about 10 to about 35, about 10 to about 40, about 10 to about45, about 15 to about 20, about 15 to about 25, about 15 to about 30,about 15 to about 35, about 15 to about 40, about 15 to about 45, about20 to about 25, about 20 to about 30, about 20 to about 35, about 20 toabout 40, about 20 to about 45, about 25 to about 30, about 25 to about35, about 25 to about 40, about 25 to about 45, about 30 to about 35,about 30 to about 40, about 30 to about 45, about 35 to about 40, about35 to about 45, about 40 to about 45, or about 45 to about 50 chemicallymodified nucleotides. In some embodiments, the chemically modified gRNAsmay comprise a total of about 1, about 3, about 5, about 10, about 15,about 20, about 25, about 30, about 35, about 40, about 45, or about 50chemically modified nucleotides. In some embodiments, the chemicallymodified gRNAs may comprise a total of at least about 1, about 3, about5, about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 45, or about 50 chemically modified nucleotides. In someembodiments, the chemically modified gRNAs may comprise a total of atmost about 3, about 5, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, or about 50 chemically modifiednucleotides. In some embodiments, the chemically modified gRNAs maycomprise a total of about 50 to about 140 chemically modifiednucleotides. In some embodiments, the chemically modified gRNAs maycomprise a total of about 50 to about 60, about 50 to about 70, about 50to about 80, about 50 to about 90, about 50 to about 100, about 50 toabout 110, about 50 to about 120, about 50 to about 130, about 50 toabout 140, about 60 to about 70, about 60 to about 80, about 60 to about90, about 60 to about 100, about 60 to about 110, about 60 to about 120,about 60 to about 130, about 60 to about 140, about 70 to about 80,about 70 to about 90, about 70 to about 100, about 70 to about 110,about 70 to about 120, about 70 to about 130, about 70 to about 140,about 80 to about 90, about 80 to about 100, about 80 to about 110,about 80 to about 120, about 80 to about 130, about 80 to about 140,about 90 to about 100, about 90 to about 110, about 90 to about 120,about 90 to about 130, about 90 to about 140, about 100 to about 110,about 100 to about 120, about 100 to about 130, about 100 to about 140,about 110 to about 120, about 110 to about 130, about 110 to about 140,about 120 to about 130, about 120 to about 140, or about 130 to about140 chemically modified nucleotides. In some embodiments, the chemicallymodified gRNAs may comprise a total of about 50, about 60, about 70,about 80, about 90, about 100, about 110, about 120, about 130, or about140 chemically modified nucleotides. In some embodiments, the chemicallymodified gRNAs may comprise a total of at least about 50, about 60,about 70, about 80, about 90, about 100, about 110, about 120, or about130 chemically modified nucleotides. In some embodiments, the chemicallymodified gRNAs may comprise a total of at most about 60, about 70, about80, about 90, about 100, about 110, about 120, about 130, or about 140chemically modified nucleotides. In one embodiment, the chemicallymodified gRNAs may comprise a total of 54 chemically modifiednucleotides. In another embodiment, the chemically modified gRNAs maycomprise a total of 62 chemically modified nucleotides.

In some embodiments, the chemically modified gRNAs may comprise about 1%to about 100% chemically modified nucleotides. In some embodiments, thechemically modified gRNAs may comprise about 1% to about 10%, about 1%to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% toabout 50%, about 1% to about 60%, about 1% to about 70%, about 1% toabout 80%, about 1% to about 90%, about 1% to about 95%, about 1% toabout 100%, about 10% to about 20%, about 10% to about 30%, about 10% toabout 40%, about 10% to about 50%, about 10% to about 60%, about 10% toabout 70%, about 10% to about 80%, about 10% to about 90%, about 10% toabout 95%, about 10% to about 100%, about 20% to about 30%, about 20% toabout 40%, about 20% to about 50%, about 20% to about 60%, about 20% toabout 70%, about 20% to about 80%, about 20% to about 90%, about 20% toabout 95%, about 20% to about 100%, about 30% to about 40%, about 30% toabout 50%, about 30% to about 60%, about 30% to about 70%, about 30% toabout 80%, about 30% to about 90%, about 30% to about 95%, about 30% toabout 100%, about 40% to about 50%, about 40% to about 60%, about 40% toabout 70%, about 40% to about 80%, about 40% to about 90%, about 40% toabout 95%, about 40% to about 100%, about 50% to about 60%, about 50% toabout 70%, about 50% to about 80%, about 50% to about 90%, about 50% toabout 95%, about 50% to about 100%, about 60% to about 70%, about 60% toabout 80%, about 60% to about 90%, about 60% to about 95%, about 60% toabout 100%, about 70% to about 80%, about 70% to about 90%, about 70% toabout 95%, about 70% to about 100%, about 80% to about 90%, about 80% toabout 95%, about 80% to about 100%, about 90% to about 95%, about 90% toabout 100%, or about 95% to about 100% chemically modified nucleotides.In some embodiments, the chemically modified gRNAs may comprise about1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, or about 100% chemicallymodified nucleotides. In some embodiments, the chemically modified gRNAsmay comprise at least about 1%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%chemically modified nucleotides. In some embodiments, the chemicallymodified gRNAs may comprise at most about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% chemically modified nucleotides.

In some embodiments, the chemically modified gRNAs may have chemicallymodified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 in the 5′-3′direction in the gRNA sequence.

In some embodiments, the chemically modified gRNAs may have chemicallymodified nucleotide on one or more of the positions which are 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairsfrom the 5′ end of the gRNA sequence.

In some embodiments, the chemically modified gRNAs may have chemicallymodified nucleotide on one or more the positions where are 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairs fromthe 3′ end of the gRNA sequence.

In some embodiments, the chemically modified gRNA may be a sgRNA. Theterm “gRNA” and “sgRNA” are used interchangeably in this application. Insome embodiments, the sgRNA may comprise one or more chemically modifiednucleotides. In some embodiments, the chemically modified sgRNA maycomprise a total of 50-150 base pairs in length. In some embodiments,the chemically modified sgRNA may comprise a total of 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, or 150 base pairs in length. In some embodiments, thechemically modified sgRNA may comprise a total of about 50 to about 140base pairs in length. In some embodiments, the chemically modified sgRNAmay comprise a total of about 50 to about 60, about 50 to about 70,about 50 to about 80, about 50 to about 90, about 50 to about 100, about50 to about 110, about 50 to about 120, about 50 to about 130, about 50to about 140, about 60 to about 70, about 60 to about 80, about 60 toabout 90, about 60 to about 100, about 60 to about 110, about 60 toabout 120, about 60 to about 130, about 60 to about 140, about 70 toabout 80, about 70 to about 90, about 70 to about 100, about 70 to about110, about 70 to about 120, about 70 to about 130, about 70 to about140, about 80 to about 90, about 80 to about 100, about 80 to about 110,about 80 to about 120, about 80 to about 130, about 80 to about 140,about 90 to about 100, about 90 to about 110, about 90 to about 120,about 90 to about 130, about 90 to about 140, about 100 to about 110,about 100 to about 120, about 100 to about 130, about 100 to about 140,about 110 to about 120, about 110 to about 130, about 110 to about 140,about 120 to about 130, about 120 to about 140, or about 130 to about140 base pairs in length. In some embodiments, the chemically modifiedsgRNAs may comprise a total of about 50, about 60, about 70, about 80,about 90, about 100, about 110, about 120, about 130, or about 140 basepairs in length. In some embodiments, the chemically modified sgRNAs maycomprise a total of at least about 50, about 60, about 70, about 80,about 90, about 100, about 110, about 120, or about 130 base pairs inlength. In some embodiments, the chemically modified sgRNA may comprisea total of at most about 60, about 70, about 80, about 90, about 100,about 110, about 120, about 130, or about 140 base pairs in length. Inone embodiment, the chemically modified sgRNA may comprise a total of100 base pairs in length. In another embodiment, the chemically modifiedsgRNA may comprise a total of 103 base pairs in length.

In some embodiments, the chemically modified sgRNAs may comprise a totalof from 1 to 150 chemically modified nucleotides. In some embodiments,the chemically modified sgRNAs may comprise a total of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 chemically modifiednucleotides. In some embodiments, the chemically modified sgRNAs maycomprise a total of about 1 to about 45 chemically modified nucleotides.In some embodiments, the chemically modified sgRNAs may comprise a totalof about 1 to about 3, about 1 to about 5, about 1 to about 10, about 1to about 15, about 1 to about 20, about 1 to about 25, about 1 to about30, about 1 to about 35, about 1 to about 40, about 1 to about 45, about3 to about 5, about 3 to about 10, about 3 to about 15, about 3 to about20, about 3 to about 25, about 3 to about 30, about 3 to about 35, about3 to about 40, about 3 to about 45, about 5 to about 10, about 5 toabout 15, about 5 to about 20, about 5 to about 25, about 5 to about 30,about 5 to about 35, about 5 to about 40, about 5 to about 45, about 10to about 15, about 10 to about 20, about 10 to about 25, about 10 toabout 30, about 10 to about 35, about 10 to about 40, about 10 to about45, about 15 to about 20, about 15 to about 25, about 15 to about 30,about 15 to about 35, about 15 to about 40, about 15 to about 45, about20 to about 25, about 20 to about 30, about 20 to about 35, about 20 toabout 40, about 20 to about 45, about 25 to about 30, about 25 to about35, about 25 to about 40, about 25 to about 45, about 30 to about 35,about 30 to about 40, about 30 to about 45, about 35 to about 40, about35 to about 45, about 40 to about 45, or about 45 to about 50 chemicallymodified nucleotides. In some embodiments, the chemically modifiedsgRNAs may comprise a total of about 1, about 3, about 5, about 10,about 15, about 20, about 25, about 30, about 35, about 40, about 45, orabout 50 chemically modified nucleotides. In some embodiments, thechemically modified sgRNAs may comprise a total of at least about 1,about 3, about 5, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, or about 50 chemically modifiednucleotides. In some embodiments, the chemically modified sgRNAs maycomprise a total of at most about 3, about 5, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, or about 50chemically modified nucleotides. In some embodiments, the chemicallymodified sgRNAs may comprise a total of about 50 to about 140 chemicallymodified nucleotides. In some embodiments, the chemically modifiedsgRNAs may comprise a total of about 50 to about 60, about 50 to about70, about 50 to about 80, about 50 to about 90, about 50 to about 100,about 50 to about 110, about 50 to about 120, about 50 to about 130,about 50 to about 140, about 60 to about 70, about 60 to about 80, about60 to about 90, about 60 to about 100, about 60 to about 110, about 60to about 120, about 60 to about 130, about 60 to about 140, about 70 toabout 80, about 70 to about 90, about 70 to about 100, about 70 to about110, about 70 to about 120, about 70 to about 130, about 70 to about140, about 80 to about 90, about 80 to about 100, about 80 to about 110,about 80 to about 120, about 80 to about 130, about 80 to about 140,about 90 to about 100, about 90 to about 110, about 90 to about 120,about 90 to about 130, about 90 to about 140, about 100 to about 110,about 100 to about 120, about 100 to about 130, about 100 to about 140,about 110 to about 120, about 110 to about 130, about 110 to about 140,about 120 to about 130, about 120 to about 140, or about 130 to about140 chemically modified nucleotides. In some embodiments, the chemicallymodified sgRNAs may comprise a total of about 50, about 60, about 70,about 80, about 90, about 100, about 110, about 120, about 130, or about140 chemically modified nucleotides. In some embodiments, the chemicallymodified sgRNAs may comprise a total of at least about 50, about 60,about 70, about 80, about 90, about 100, about 110, about 120, or about130 chemically modified nucleotides. In some embodiments, the chemicallymodified sgRNAs may comprise a total of at most about 60, about 70,about 80, about 90, about 100, about 110, about 120, about 130, or about140 chemically modified nucleotides. In one embodiment, the chemicallymodified sgRNAs may comprise a total of 54 chemically modifiednucleotides. In another embodiment, the chemically modified sgRNAs maycomprise a total of 62 chemically modified nucleotides.

In some embodiments, the chemically modified sgRNAs may comprise about1% to about 100% chemically modified nucleotides. In some embodiments,the chemically modified sgRNAs may comprise about 1% to about 10%, about1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1%to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% toabout 80%, about 1% to about 90%, about 1% to about 95%, about 1% toabout 100%, about 10% to about 20%, about 10% to about 30%, about 10% toabout 40%, about 10% to about 50%, about 10% to about 60%, about 10% toabout 70%, about 10% to about 80%, about 10% to about 90%, about 10% toabout 95%, about 10% to about 100%, about 20% to about 30%, about 20% toabout 40%, about 20% to about 50%, about 20% to about 60%, about 20% toabout 70%, about 20% to about 80%, about 20% to about 90%, about 20% toabout 95%, about 20% to about 100%, about 30% to about 40%, about 30% toabout 50%, about 30% to about 60%, about 30% to about 70%, about 30% toabout 80%, about 30% to about 90%, about 30% to about 95%, about 30% toabout 100%, about 40% to about 50%, about 40% to about 60%, about 40% toabout 70%, about 40% to about 80%, about 40% to about 90%, about 40% toabout 95%, about 40% to about 100%, about 50% to about 60%, about 50% toabout 70%, about 50% to about 80%, about 50% to about 90%, about 50% toabout 95%, about 50% to about 100%, about 60% to about 70%, about 60% toabout 80%, about 60% to about 90%, about 60% to about 95%, about 60% toabout 100%, about 70% to about 80%, about 70% to about 90%, about 70% toabout 95%, about 70% to about 100%, about 80% to about 90%, about 80% toabout 95%, about 80% to about 100%, about 90% to about 95%, about 90% toabout 100%, or about 95% to about 100% chemically modified nucleotides.In some embodiments, the chemically modified sgRNAs may comprise about1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, or about 100% chemicallymodified nucleotides. In some embodiments, the chemically modifiedsgRNAs may comprise at least about 1%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 95% chemically modified nucleotides. In some embodiments, thechemically modified sgRNAs may comprise at most about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, or about 100% chemically modified nucleotides.

In some embodiments, the chemically modified sgRNAs may have chemicallymodified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 in the 5′-3′direction in the sgRNA sequence.

In some embodiments, the chemically modified sgRNAs may have chemicallymodified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairs from the 5′end of the sgRNA sequence.

In some embodiments, the chemically modified sgRNAs may have chemicallymodified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, or 149 base pairs from the 3′end of the sgRNA sequence.

In some embodiments, the chemically modified gRNAs or sgRNAs may haveone or more chemical modifications in the spacer or protospacer region,i.e., the target sequence. In some embodiments, the chemically modifiedgRNAs or sgRNAs may comprise a spacer sequence of from 10 to 30 basepairs in length. In some embodiments, the chemically modified gRNAs orsgRNAs may comprise a spacer sequence of 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs inlength. In some embodiments, the chemically modified gRNAs or sgRNAs maycomprise a spacer sequence of about 10 to about 30 base pairs in length.In some embodiments, the chemically modified gRNAs or sgRNAs maycomprise a spacer sequence of about 10 to about 15, about 10 to about20, about 10 to about 25, about 10 to about 30, about 15 to about 20,about 15 to about 25, about 15 to about 30, about 20 to about 25, about20 to about 30, or about 25 to about 30 base pairs in length. In someembodiments, the chemically modified gRNAs or sgRNAs may comprise aspacer sequence of about 10, about 15, about 20, about 25, or about 30base pairs in length. In some embodiments, the chemically modified gRNAsor sgRNAs may comprise a spacer sequence of at least about 10, about 15,about 20, or about 25 base pairs in length. In some embodiments, thechemically modified gRNAs or sgRNAs may comprise a spacer sequence of atmost about 15, about 20, about 25, or about 30 base pairs in length. Inone embodiment, the chemically modified gRNAs or sgRNAs may comprise aspacer sequence of 18 base pairs in length. In another embodiment, thechemically modified gRNAs or sgRNAs may comprise a spacer sequence of 22base pairs in length. In a preferred embodiment, the chemically modifiedgRNAs or sgRNAs may comprise a spacer sequence of 20 base pairs inlength.

In some embodiments, the spacer sequence may comprise a total of from 1to 30 chemically modified nucleotides. In some embodiments, the spacerregion may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 chemically modified nucleotides. In some embodiments, the spacersequence may comprise a total of about 1 to about 10 chemically modifiednucleotides. In some embodiments, the spacer sequence may comprise atotal of about 1 to about 2, about 1 to about 3, about 1 to about 4,about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 toabout 8, about 1 to about 9, about 1 to about 10, about 2 to about 3,about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 toabout 7, about 2 to about 8, about 2 to about 9, about 2 to about 10,about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 toabout 7, about 3 to about 8, about 3 to about 9, about 3 to about 10,about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 toabout 8, about 4 to about 9, about 4 to about 10, about 5 to about 6,about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 toabout 10, about 6 to about 7, about 6 to about 8, about 6 to about 9,about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 toabout 10, about 8 to about 9, about 8 to about 10, or about 9 to about10 chemically modified nucleotides. In some embodiments, the spacersequence may comprise a total of about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, or about 10 chemicallymodified nucleotides. In some embodiments, the spacer sequence maycomprise a total of at least about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, or about 9 chemically modifiednucleotides. In some embodiments, the spacer sequence may comprise atotal of at most about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, or about 10 chemically modified nucleotides. In someembodiments, the spacer sequence may comprise a total of about 10 toabout 30 chemically modified nucleotides. In some embodiments, thespacer sequence may comprise a total of about 10 to about 15, about 10to about 20, about 10 to about 25, about 10 to about 30, about 15 toabout 20, about 15 to about 25, about 15 to about 30, about 20 to about25, about 20 to about 30, or about 25 to about 30 chemically modifiednucleotides. In some embodiments, the spacer sequence may comprise atotal of about 10, about 15, about 20, about 25, or about 30 chemicallymodified nucleotides. In some embodiments, the spacer sequence maycomprise a total of at least about 10, about 15, about 20, or about 25chemically modified nucleotides. In some embodiments, the spacersequence may comprise a total of at most about 15, about 20, about 25,or about 30 chemically modified nucleotides. In one embodiment, thespacer sequence may comprise a total of 3 chemically modifiednucleotides. In another embodiment, the spacer sequence may comprise atotal of 5 chemically modified nucleotides.

In some embodiments, the spacer sequence may comprise from 1% to 100%chemically modified nucleotides. In some embodiments, the spacersequence may comprise about 1% to about 100% chemically modifiednucleotides. In some embodiments, the spacer sequence may comprise about1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1%to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% toabout 70%, about 1% to about 80%, about 1% to about 90%, about 1% toabout 95%, about 1% to about 100%, about 10% to about 20%, about 10% toabout 30%, about 10% to about 40%, about 10% to about 50%, about 10% toabout 60%, about 10% to about 70%, about 10% to about 80%, about 10% toabout 90%, about 10% to about 95%, about 10% to about 100%, about 20% toabout 30%, about 20% to about 40%, about 20% to about 50%, about 20% toabout 60%, about 20% to about 70%, about 20% to about 80%, about 20% toabout 90%, about 20% to about 95%, about 20% to about 100%, about 30% toabout 40%, about 30% to about 50%, about 30% to about 60%, about 30% toabout 70%, about 30% to about 80%, about 30% to about 90%, about 30% toabout 95%, about 30% to about 100%, about 40% to about 50%, about 40% toabout 60%, about 40% to about 70%, about 40% to about 80%, about 40% toabout 90%, about 40% to about 95%, about 40% to about 100%, about 50% toabout 60%, about 50% to about 70%, about 50% to about 80%, about 50% toabout 90%, about 50% to about 95%, about 50% to about 100%, about 60% toabout 70%, about 60% to about 80%, about 60% to about 90%, about 60% toabout 95%, about 60% to about 100%, about 70% to about 80%, about 70% toabout 90%, about 70% to about 95%, about 70% to about 100%, about 80% toabout 90%, about 80% to about 95%, about 80% to about 100%, about 90% toabout 95%, about 90% to about 100%, or about 95% to about 100%chemically modified nucleotides. In some embodiments, the spacersequence may comprise about 1%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,or about 100% chemically modified nucleotides. In some embodiments, thespacer sequence may comprise at least about 1%, about 10%, about 200%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or about 95% chemically modified nucleotides. In some embodiments,the spacer sequence may comprise at most about 10%, about 20%, about30%, about 40%, about 50%, about 600%, about 70%, about 80%, about 90%,about 95%, or about 100% chemically modified nucleotides.

In some embodiments, the chemically modified spacer sequence may havechemically modified nucleotide on one or more of the positions 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 in the 5′-3′ direction in the spacersequence.

In some embodiments, the chemically modified spacer region may havechemically modified nucleotide on one or more of the positions which are1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, or 29 base pairs from the 5′ end ofthespacer sequence.

In some embodiments, the chemically modified spacer region may havechemically modified nucleotide on one or more the positions which are 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, or 29 base pairs from the 3′ end of thespacer sequence.

In some embodiments, the chemically modified gRNAs or sgRNAs may haveone or more chemical modifications in the tracrRNA sequence. In someembodiments, the chemically modified gRNAs or sgRNAs may comprise atracrRNA sequence of from 30 to 100 base pairs in length. In someembodiments, the chemically modified gRNAs or sgRNAs may comprise atracrRNA sequence of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, or 130 base pairs in length. In some embodiments,the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequenceof from about 30 to about 130 base pairs in length. In some embodiments,the chemically modified gRNAs or sgRNAs may comprise a tracrRNA sequenceof from about 30 to about 40, about 30 to about 50, about 30 to about60, about 30 to about 70, about 30 to about 75, about 30 to about 80,about 30 to about 90, about 30 to about 100, about 30 to about 110,about 30 to about 120, about 30 to about 130, about 40 to about 50,about 40 to about 60, about 40 to about 70, about 40 to about 75, about40 to about 80, about 40 to about 90, about 40 to about 100, about 40 toabout 110, about 40 to about 120, about 40 to about 130, about 50 toabout 60, about 50 to about 70, about 50 to about 75, about 50 to about80, about 50 to about 90, about 50 to about 100, about 50 to about 110,about 50 to about 120, about 50 to about 130, about 60 to about 70,about 60 to about 75, about 60 to about 80, about 60 to about 90, about60 to about 100, about 60 to about 110, about 60 to about 120, about 60to about 130, about 70 to about 75, about 70 to about 80, about 70 toabout 90, about 70 to about 100, about 70 to about 110, about 70 toabout 120, about 70 to about 130, about 75 to about 80, about 75 toabout 90, about 75 to about 100, about 75 to about 110, about 75 toabout 120, about 75 to about 130, about 80 to about 90, about 80 toabout 100, about 80 to about 110, about 80 to about 120, about 80 toabout 130, about 90 to about 100, about 90 to about 110, about 90 toabout 120, about 90 to about 130, about 100 to about 110, about 100 toabout 120, about 100 to about 130, about 110 to about 120, about 110 toabout 130, or about 120 to about 130 base pairs in length. In someembodiments, the chemically modified gRNAs or sgRNAs may comprise atracrRNA sequence of from about 30, about 40, about 50, about 60, about70, about 75, about 80, about 90, about 100, about 110, about 120, orabout 130 base pairs in length. In some embodiments, the chemicallymodified gRNAs or sgRNAs may comprise a tracrRNA sequence of from atleast about 30, about 40, about 50, about 60, about 70, about 75, about80, about 90, about 100, about 110, or about 120 base pairs in length.In some embodiments, the chemically modified gRNAs or sgRNAs maycomprise a tracrRNA sequence of from at most about 40, about 50, about60, about 70, about 75, about 80, about 90, about 100, about 110, about120, or about 130 base pairs in length. In some embodiments, thechemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence of70 base pairs in length. In some embodiments, the chemically modifiedgRNAs or sgRNAs may comprise a tracrRNA sequence of 73 base pairs inlength. In some embodiments, the chemically modified gRNAs or sgRNAs maycomprise a tracrRNA sequence of 68 base pairs in length. In someembodiments, the chemically modified gRNAs or sgRNAs may comprise atracrRNA sequence of 71 base pairs in length.

In some embodiments, the tracrRNA sequence may comprise a total of from1 to 130 chemically modified nucleotides. In some embodiments, thechemically modified gRNAs or sgRNAs may comprise a tracrRNA sequence offrom 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, or 130 chemically modifiednucleotides. In some embodiments, the tracrRNA sequence may comprise atotal of from about 1 to about 10 chemically modified nucleotides. Insome embodiments, the tracrRNA sequence may comprise a total of fromabout 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 toabout 5, about 1 to about 6, about 1 to about 7, about 1 to about 8,about 1 to about 9, about 1 to about 10, about 2 to about 3, about 2 toabout 4, about 2 to about 5, about 2 to about 6, about 2 to about 7,about 2 to about 8, about 2 to about 9, about 2 to about 10, about 3 toabout 4, about 3 to about 5, about 3 to about 6, about 3 to about 7,about 3 to about 8, about 3 to about 9, about 3 to about 10, about 4 toabout 5, about 4 to about 6, about 4 to about 7, about 4 to about 8,about 4 to about 9, about 4 to about 10, about 5 to about 6, about 5 toabout 7, about 5 to about 8, about 5 to about 9, about 5 to about 10,about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 toabout 10, about 7 to about 8, about 7 to about 9, about 7 to about 10,about 8 to about 9, about 8 to about 10, or about 9 to about 10chemically modified nucleotides. In some embodiments, the tracrRNAsequence may comprise a total of from about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, or about 10 chemicallymodified nucleotides. In some embodiments, the tracrRNA sequence maycomprise a total of from at least about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, or about 9 chemically modifiednucleotides. In some embodiments, the tracrRNA sequence may comprise atotal of from at most about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, or about 10 chemically modified nucleotides. Insome embodiments, the tracrRNA sequence may comprise a total of fromabout 10 to about 30 chemically modified nucleotides. In someembodiments, the tracrRNA sequence may comprise a total of from about 10to about 15, about 10 to about 20, about 10 to about 25, about 10 toabout 30, about 15 to about 20, about 15 to about 25, about 15 to about30, about 20 to about 25, about 20 to about 30, or about 25 to about 30chemically modified nucleotides. In some embodiments, the tracrRNAsequence may comprise a total of from about 10, about 15, about 20,about 25, or about 30 chemically modified nucleotides. In someembodiments, the tracrRNA sequence may comprise a total of from at leastabout 10, about 15, about 20, or about 25 chemically modifiednucleotides. In some embodiments, the spacer region may comprise a totalof at most about 15, about 20, about 25, or about 30 chemically modifiednucleotides. In some embodiments, the tracrRNA sequence may comprise atotal of from about 30 to about 130 chemically modified nucleotides. Insome embodiments, the tracrRNA sequence may comprise a total of fromabout 30 to about 40, about 30 to about 50, about 30 to about 60, about30 to about 70, about 30 to about 75, about 30 to about 80, about 30 toabout 90, about 30 to about 100, about 30 to about 110, about 30 toabout 120, about 30 to about 130, about 40 to about 50, about 40 toabout 60, about 40 to about 70, about 40 to about 75, about 40 to about80, about 40 to about 90, about 40 to about 100, about 40 to about 110,about 40 to about 120, about 40 to about 130, about 50 to about 60,about 50 to about 70, about 50 to about 75, about 50 to about 80, about50 to about 90, about 50 to about 100, about 50 to about 110, about 50to about 120, about 50 to about 130, about 60 to about 70, about 60 toabout 75, about 60 to about 80, about 60 to about 90, about 60 to about100, about 60 to about 110, about 60 to about 120, about 60 to about130, about 70 to about 75, about 70 to about 80, about 70 to about 90,about 70 to about 100, about 70 to about 110, about 70 to about 120,about 70 to about 130, about 75 to about 80, about 75 to about 90, about75 to about 100, about 75 to about 110, about 75 to about 120, about 75to about 130, about 80 to about 90, about 80 to about 100, about 80 toabout 110, about 80 to about 120, about 80 to about 130, about 90 toabout 100, about 90 to about 110, about 90 to about 120, about 90 toabout 130, about 100 to about 110, about 100 to about 120, about 100 toabout 130, about 110 to about 120, about 110 to about 130, or about 120to about 130 chemically modified nucleotides. In some embodiments, thetracrRNA sequence may comprise a total of from about 30, about 40, about50, about 60, about 70, about 75, about 80, about 90, about 100, about110, about 120, or about 130 chemically modified nucleotides. In someembodiments, the tracrRNA sequence may comprise a total of from at leastabout 30, about 40, about 50, about 60, about 70, about 75, about 80,about 90, about 100, about 110, or about 120 chemically modifiednucleotides. In some embodiments, the tracrRNA sequence may comprise atotal of from at most about 40, about 50, about 60, about 70, about 75,about 80, about 90, about 100, about 110, about 120, or about 130chemically modified nucleotides. In one embodiment, the tracrRNAsequence may comprise a total of 51 chemically modified nucleotides. Inanother embodiment, the tracrRNA sequence may comprise a total of 59chemically modified nucleotides.

In some embodiments, the tracrRNA sequence may comprise from 1% to 100%chemically modified nucleotides. In some embodiments, the tracrRNAsequence may comprise about 1% to about 100% chemically modifiednucleotides. In some embodiments, the tracrRNA sequence may compriseabout 1% to about 10%, about 1% to about 20%, about 1% to about 30%,about 1% to about 40%, about 1% to about 50%, about 1% to about 60%,about 1% to about 70%, about 1% to about 80%, about 1% to about 90%,about 1% to about 95%, about 1% to about 100%, about 10% to about 20%,about 10% to about 30%, about 10% to about 40%, about 10% to about 50%,about 10% to about 60%, about 10% to about 70%, about 10% to about 80%,about 10% to about 90%, about 10% to about 95%, about 10% to about 100%,about 20% to about 30%, about 20% to about 40%, about 20% to about 50%,about 20% to about 60%, about 20% to about 70%, about 20% to about 80%,about 20% to about 90%, about 20% to about 95%, about 20% to about 100%,about 30% to about 40%, about 30% to about 50%, about 30% to about 60%,about 30% to about 70%, about 30% to about 80%, about 30% to about 90%,about 30% to about 95%, about 30% to about 100%, about 40% to about 50%,about 40% to about 60%, about 40% to about 70%, about 40% to about 80%,about 40% to about 90%, about 40% to about 95%, about 40% to about 100%,about 50% to about 60%, about 50% to about 70%, about 50% to about 80%,about 50% to about 90%, about 50% to about 95%, about 50% to about 100%,about 60% to about 70%, about 60% to about 80%, about 60% to about 90%,about 60% to about 95%, about 60% to about 100%, about 70% to about 80%,about 70% to about 90%, about 70% to about 95%, about 70% to about 100%,about 80% to about 90%, about 80% to about 95%, about 80% to about 100%,about 90% to about 95%, about 90% to about 100%, or about 95% to about100% chemically modified nucleotides. In some embodiments, the tracrRNAsequence may comprise about 1%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,or about 100% chemically modified nucleotides. In some embodiments, thetracrRNA sequence may comprise at least about 1%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or about 95% chemically modified nucleotides. In some embodiments,the tracrRNA sequence may comprise at most about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or about 100% chemically modified nucleotides.

In some embodiments, the chemically modified tracrRNA sequence may havechemically modified nucleotide on one or more of the positions 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, or 130 in the 5′-3′ direction in thetracrRNA sequence.

In some embodiments, the chemically modified tracr region may havechemically modified nucleotide on one or more of the positions which are1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, or 129 base pairs from the 5′ end of thetracrRNA sequence.

In some embodiments, the chemically modified tracr region may havechemically modified nucleotide on one or more the positions which are 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, or 129 base pairs from the 3′ end of thetracrRNA sequence.

In some embodiments, the chemically modified gRNAs may have one or morechemical modifications in the 5′ end of the gRNA sequence. In someembodiments, the chemically modified gRNAs may comprise one or morechemically modified nucleotides in the 5′ end of the gRNA sequence. Insome embodiments, the chemically modified gRNAs may have one or morechemical modifications in the 3′ end of the gRNA sequence. In someembodiments, the chemically modified gRNAs may comprise one or morechemically modified nucleotides in the 3′ end of the gRNA sequence.

In some embodiments, the chemically modified gRNAs may comprise a crRNA.In some embodiments, the crRNA may comprise one or more chemicallymodified nucleotides. In some embodiments, the chemically modified crRNAmay be of from 30 to 50 base pairs in length. In some embodiments, thechemically modified crRNA may be of 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs in length.In some embodiments, the chemically modified crRNA may be of from about30 to about 50 base pairs in length. In some embodiments, the chemicallymodified crRNA may be of from about 30 to about 32, about 30 to about34, about 30 to about 36, about 30 to about 38, about 30 to about 40,about 30 to about 42, about 30 to about 44, about 30 to about 46, about30 to about 48, about 30 to about 50, about 32 to about 34, about 32 toabout 36, about 32 to about 38, about 32 to about 40, about 32 to about42, about 32 to about 44, about 32 to about 46, about 32 to about 48,about 32 to about 50, about 34 to about 36, about 34 to about 38, about34 to about 40, about 34 to about 42, about 34 to about 44, about 34 toabout 46, about 34 to about 48, about 34 to about 50, about 36 to about38, about 36 to about 40, about 36 to about 42, about 36 to about 44,about 36 to about 46, about 36 to about 48, about 36 to about 50, about38 to about 40, about 38 to about 42, about 38 to about 44, about 38 toabout 46, about 38 to about 48, about 38 to about 50, about 40 to about42, about 40 to about 44, about 40 to about 46, about 40 to about 48,about 40 to about 50, about 42 to about 44, about 42 to about 46, about42 to about 48, about 42 to about 50, about 44 to about 46, about 44 toabout 48, about 44 to about 50, about 46 to about 48, about 46 to about50, or about 48 to about 50 base pairs in length. In some embodiments,the chemically modified crRNA may be of from about 30, about 32, about34, about 36, about 38, about 40, about 42, about 44, about 46, about48, or about 50 base pairs in length. In some embodiments, thechemically modified crRNA may be of from at least about 30, about 32,about 34, about 36, about 38, about 40, about 42, about 44, about 46, orabout 48 base pairs in length. In some embodiments, the chemicallymodified crRNA may be of from at most about 32, about 34, about 36,about 38, about 40, about 42, about 44, about 46, about 48, or about 50base pairs in length.

In some embodiments, the crRNA may comprise a total of from 1 to 50chemically modified nucleotides. In some embodiments, the chemicallymodified crRNA may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 chemically modified nucleotides. In some embodiments,the chemically modified crRNA may comprise a total of about 1 to about10 chemically modified nucleotides. In some embodiments, the chemicallymodified crRNA may comprise a total of about 1 to about 2, about 1 toabout 3, about 1 to about 4, about 1 to about 5, about 1 to about 6,about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 toabout 10, about 2 to about 3, about 2 to about 4, about 2 to about 5,about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 toabout 9, about 2 to about 10, about 3 to about 4, about 3 to about 5,about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 toabout 9, about 3 to about 10, about 4 to about 5, about 4 to about 6,about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 toabout 10, about 5 to about 6, about 5 to about 7, about 5 to about 8,about 5 to about 9, about 5 to about 10, about 6 to about 7, about 6 toabout 8, about 6 to about 9, about 6 to about 10, about 7 to about 8,about 7 to about 9, about 7 to about 10, about 8 to about 9, about 8 toabout 10, or about 9 to about 10 chemically modified nucleotides. Insome embodiments, the chemically modified crRNA may comprise a total ofabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, or about 10 chemically modified nucleotides. In someembodiments, the chemically modified crRNA may comprise a total of atleast about 1, about 2, about 3, about 4, about 5, about 6, about 7,about 8, or about 9 chemically modified nucleotides. In someembodiments, the chemically modified crRNA may comprise a total of atmost about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, or about 10 chemically modified nucleotides. In someembodiments, the chemically modified crRNA may comprise a total of about10 to about 30 chemically modified nucleotides. In some embodiments, thechemically modified crRNA may comprise a total of about 10 to about 15,about 10 to about 20, about 10 to about 25, about 10 to about 30, about15 to about 20, about 15 to about 25, about 15 to about 30, about 20 toabout 25, about 20 to about 30, or about 25 to about 30 chemicallymodified nucleotides. In some embodiments, the chemically modified crRNAmay comprise a total of about 10, about 15, about 20, about 25, or about30 chemically modified nucleotides. In some embodiments, the chemicallymodified crRNA may comprise a total of at least about 10, about 15,about 20, or about 25 chemically modified nucleotides. In someembodiments, the chemically modified crRNA may comprise a total of atmost about 15, about 20, about 25, or about 30 chemically modifiednucleotides. In some embodiments, the chemically modified crRNA maycomprise a total of from about 30 to about 50 chemically modifiednucleotides. In some embodiments, the chemically modified crRNA maycomprise a total of from about 30 to about 32, about 30 to about 34,about 30 to about 36, about 30 to about 38, about 30 to about 40, about30 to about 42, about 30 to about 44, about 30 to about 46, about 30 toabout 48, about 30 to about 50, about 32 to about 34, about 32 to about36, about 32 to about 38, about 32 to about 40, about 32 to about 42,about 32 to about 44, about 32 to about 46, about 32 to about 48, about32 to about 50, about 34 to about 36, about 34 to about 38, about 34 toabout 40, about 34 to about 42, about 34 to about 44, about 34 to about46, about 34 to about 48, about 34 to about 50, about 36 to about 38,about 36 to about 40, about 36 to about 42, about 36 to about 44, about36 to about 46, about 36 to about 48, about 36 to about 50, about 38 toabout 40, about 38 to about 42, about 38 to about 44, about 38 to about46, about 38 to about 48, about 38 to about 50, about 40 to about 42,about 40 to about 44, about 40 to about 46, about 40 to about 48, about40 to about 50, about 42 to about 44, about 42 to about 46, about 42 toabout 48, about 42 to about 50, about 44 to about 46, about 44 to about48, about 44 to about 50, about 46 to about 48, about 46 to about 50, orabout 48 to about 50 chemically modified nucleotides. In someembodiments, the chemically modified crRNA may comprise a total of fromabout 30, about 32, about 34, about 36, about 38, about 40, about 42,about 44, about 46, about 48, or about 50 chemically modifiednucleotides. In some embodiments, the chemically modified crRNA maycomprise a total of from at least about 30, about 32, about 34, about36, about 38, about 40, about 42, about 44, about 46, or about 48chemically modified nucleotides. In some embodiments, the chemicallymodified crRNA may comprise a total of from at most about 32, about 34,about 36, about 38, about 40, about 42, about 44, about 46, about 48, orabout 50 chemically modified nucleotides.

In some embodiments, the crRNA may comprise from 1% to 100% chemicallymodified nucleotides. In some embodiments, the crRNA may comprise about1% to about 100% chemically modified nucleotides. In some embodiments,the crRNA may comprise about 1% to about 10%, about 1% to about 20%,about 1% to about 30%, about 1% to about 40%, about 1% to about 50%,about 1% to about 60%, about 1% to about 70%, about 1% to about 80%,about 1% to about 90%, about 1% to about 95%, about 1% to about 100%,about 10% to about 20%, about 10% to about 30%, about 10% to about 40%,about 10% to about 50%, about 10% to about 60%, about 10% to about 70%,about 10% to about 80%, about 10% to about 90%, about 10% to about 95%,about 10% to about 100%, about 20% to about 30%, about 20% to about 40%,about 20% to about 50%, about 20% to about 60%, about 20% to about 70%,about 20% to about 80%, about 20% to about 90%, about 20% to about 95%,about 20% to about 100%, about 30% to about 40%, about 30% to about 50%,about 30% to about 60%, about 30% to about 70%, about 30% to about 80%,about 30% to about 90%, about 30% to about 95%, about 30% to about 100%,about 40% to about 50%, about 40% to about 60%, about 40% to about 70%,about 40% to about 80%, about 40% to about 90%, about 40% to about 95%,about 40% to about 100%, about 50% to about 60%, about 50% to about 70%,about 50% to about 80%, about 50% to about 90%, about 50% to about 95%,about 50% to about 100%, about 60% to about 70%, about 60% to about 80%,about 60% to about 90%, about 60% to about 95%, about 60% to about 100%,about 70% to about 80%, about 70% to about 90%, about 70% to about 95%,about 70% to about 100%, about 80% to about 90%, about 80% to about 95%,about 80% to about 100%, about 90% to about 95%, about 90% to about100%, or about 95% to about 100% chemically modified nucleotides. Insome embodiments, the crRNA may comprise about 1%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, or about 100% chemically modified nucleotides. In someembodiments, the crRNA may comprise at least about 1%, about 10%, about20%, about 30%, about 40%, about 500%, about 60%, about 70%, about 80%,about 90%, or about 95% chemically modified nucleotides. In someembodiments, the crRNA may comprise at most about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%about 95%, or about 100% chemically modified nucleotides.

In some embodiments, the chemically modified crRNA may have chemicallymodified nucleotide on one or more of the positions 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 in the 5′-3′ direction in the crRNAsequence.

In some embodiments, the chemically modified crRNA may have chemicallymodified nucleotide on one or more of the positions which are 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs from the 5′ end of thecrRNA sequence.

In some embodiments, the chemically modified crRNA may have chemicallymodified nucleotide on one or more of the positions which are 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs from the 3′ end of thecrRNA sequence.

In some embodiments, the chemically modified gRNAs may comprise atracrRNA. In some embodiments, the tracrRNA may comprise one or morechemically modified nucleotides. In some embodiments, the chemicallymodified tracrRNA may comprise a total of from 50 to 130 base pairs inlength. In some embodiments, the chemically modified tracrRNA maycomprise a total of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, or 130 base pairs in length. In some embodiments,the chemically modified tracrRNA may comprise a total of about 30 toabout 130 base pairs in length. In some embodiments, the chemicallymodified tracrRNA may comprise a total of about 30 to about 40, about 30to about 50, about 30 to about 60, about 30 to about 70, about 30 toabout 75, about 30 to about 80, about 30 to about 90, about 30 to about100, about 30 to about 110, about 30 to about 120, about 30 to about130, about 40 to about 50, about 40 to about 60, about 40 to about 70,about 40 to about 75, about 40 to about 80, about 40 to about 90, about40 to about 100, about 40 to about 110, about 40 to about 120, about 40to about 130, about 50 to about 60, about 50 to about 70, about 50 toabout 75, about 50 to about 80, about 50 to about 90, about 50 to about100, about 50 to about 110, about 50 to about 120, about 50 to about130, about 60 to about 70, about 60 to about 75, about 60 to about 80,about 60 to about 90, about 60 to about 100, about 60 to about 110,about 60 to about 120, about 60 to about 130, about 70 to about 75,about 70 to about 80, about 70 to about 90, about 70 to about 100, about70 to about 110, about 70 to about 120, about 70 to about 130, about 75to about 80, about 75 to about 90, about 75 to about 100, about 75 toabout 110, about 75 to about 120, about 75 to about 130, about 80 toabout 90, about 80 to about 100, about 80 to about 110, about 80 toabout 120, about 80 to about 130, about 90 to about 100, about 90 toabout 110, about 90 to about 120, about 90 to about 130, about 100 toabout 110, about 100 to about 120, about 100 to about 130, about 110 toabout 120, about 110 to about 130, or about 120 to about 130 base pairsin length. In some embodiments, the chemically modified tracrRNA maycomprise a total of about 30, about 40, about 50, about 60, about 70,about 75, about 80, about 90, about 100, about 110, about 120, or about130 base pairs in length. In some embodiments, the chemically modifiedtracrRNA may comprise a total of at least about 30, about 40, about 50,about 60, about 70, about 75, about 80, about 90, about 100, about 110,or about 120 base pairs in length. In some embodiments, the chemicallymodified tracrRNA may comprise a total of at most about 40, about 50,about 60, about 70, about 75, about 80, about 90, about 100, about 110,about 120, or about 130 base pairs in length.

In some embodiments, the chemically modified tracrRNA may comprise atotal of from 1 to 130 chemically modified nucleotides. In someembodiments, the chemically modified tracrRNA may comprise a total of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, or 130 chemically modifiednucleotides. In some embodiments, the chemically modified tracrRNA maycomprise a total of about 1 to about 10 chemically modified nucleotides.In some embodiments, the chemically modified tracrRNA may comprise atotal of about 1 to about 2, about 1 to about 3, about 1 to about 4,about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 toabout 8, about 1 to about 9, about 1 to about 10, about 2 to about 3,about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 toabout 7, about 2 to about 8, about 2 to about 9, about 2 to about 10,about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 toabout 7, about 3 to about 8, about 3 to about 9, about 3 to about 10,about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 toabout 8, about 4 to about 9, about 4 to about 10, about 5 to about 6,about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 toabout 10, about 6 to about 7, about 6 to about 8, about 6 to about 9,about 6 to about 10, about 7 to about 8, about 7 to about 9, about 7 toabout 10, about 8 to about 9, about 8 to about 10, or about 9 to about10 chemically modified nucleotides. In some embodiments, the chemicallymodified tracrRNA may comprise a total of about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, or about 10chemically modified nucleotides. In some embodiments, the chemicallymodified tracrRNA may comprise a total of at least about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, or about 9chemically modified nucleotides. In some embodiments, the chemicallymodified tracrRNA may comprise a total of at most about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, or about 10chemically modified nucleotides. In some embodiments, the chemicallymodified tracrRNA may comprise a total of about 10 to about 30chemically modified nucleotides. In some embodiments, the chemicallymodified tracrRNA may comprise a total of about 10 to about 15, about 10to about 20, about 10 to about 25, about 10 to about 30, about 15 toabout 20, about 15 to about 25, about 15 to about 30, about 20 to about25, about 20 to about 30, or about 25 to about 30 chemically modifiednucleotides. In some embodiments, the chemically modified tracrRNA maycomprise a total of about 10, about 15, about 20, about 25, or about 30chemically modified nucleotides. In some embodiments, the chemicallymodified tracrRNA may comprise a total of at least about 10, about 15,about 20, or about 25 chemically modified nucleotides. In someembodiments, the spacer region may comprise a total of at most about 15,about 20, about 25, or about 30 chemically modified nucleotides. In someembodiments, the chemically modified tracrRNA may comprise a total ofabout 30 to about 130 chemically modified nucleotides. In someembodiments, the chemically modified tracrRNA may comprise a total ofabout 30 to about 40, about 30 to about 50, about 30 to about 60, about30 to about 70, about 30 to about 75, about 30 to about 80, about 30 toabout 90, about 30 to about 100, about 30 to about 110, about 30 toabout 120, about 30 to about 130, about 40 to about 50, about 40 toabout 60, about 40 to about 70, about 40 to about 75, about 40 to about80, about 40 to about 90, about 40 to about 100, about 40 to about 110,about 40 to about 120, about 40 to about 130, about 50 to about 60,about 50 to about 70, about 50 to about 75, about 50 to about 80, about50 to about 90, about 50 to about 100, about 50 to about 110, about 50to about 120, about 50 to about 130, about 60 to about 70, about 60 toabout 75, about 60 to about 80, about 60 to about 90, about 60 to about100, about 60 to about 110, about 60 to about 120, about 60 to about130, about 70 to about 75, about 70 to about 80, about 70 to about 90,about 70 to about 100, about 70 to about 110, about 70 to about 120,about 70 to about 130, about 75 to about 80, about 75 to about 90, about75 to about 100, about 75 to about 110, about 75 to about 120, about 75to about 130, about 80 to about 90, about 80 to about 100, about 80 toabout 110, about 80 to about 120, about 80 to about 130, about 90 toabout 100, about 90 to about 110, about 90 to about 120, about 90 toabout 130, about 100 to about 110, about 100 to about 120, about 100 toabout 130, about 110 to about 120, about 110 to about 130, or about 120to about 130 chemically modified nucleotides. In some embodiments, thechemically modified tracrRNA may comprise a total of about 30, about 40,about 50, about 60, about 70, about 75, about 80, about 90, about 100,about 110, about 120, or about 130 chemically modified nucleotides. Insome embodiments, the chemically modified tracrRNA may comprise a totalof at least about 30, about 40, about 50, about 60, about 70, about 75,about 80, about 90, about 100, about 110, or about 120 chemicallymodified nucleotides. In some embodiments, the chemically modifiedtracrRNA may comprise a total of at most about 40, about 50, about 60,about 70, about 75, about 80, about 90, about 100, about 110, about 120,or about 130 chemically modified nucleotides.

In some embodiments, the chemically modified tracrRNA may comprise about1% to about 100% chemically modified nucleotides. In some embodiments,the chemically modified tracrRNA may comprise about 1% to about 10%,about 1% to about 20%, about 1% to about 30%, about 1% to about 40%,about 1% to about 50%, about 1% to about 60%, about 1% to about 70%,about 1% to about 80%, about 1% to about 90%, about 1% to about 95%,about 1% to about 100%, about 10% to about 20%, about 10% to about 30%,about 10% to about 40%, about 10% to about 50%, about 10% to about 60%,about 10% to about 70%, about 10% to about 80%, about 10% to about 90%,about 10% to about 95%, about 10% to about 100%, about 20% to about 30%,about 20% to about 40%, about 20% to about 50%, about 20% to about 60%,about 20% to about 70%, about 20% to about 80%, about 20% to about 90%,about 20% to about 95%, about 20% to about 100%, about 30% to about 40%,about 30% to about 50%, about 30% to about 60%, about 30% to about 70%,about 30% to about 80%, about 30% to about 90%, about 30% to about 95%,about 30% to about 100%, about 40% to about 50%, about 40% to about 60%,about 40% to about 70%, about 40% to about 80%, about 40% to about 90%,about 40% to about 95%, about 40% to about 100%, about 50% to about 60%,about 50% to about 70%, about 50% to about 80%, about 50% to about 90%,about 50% to about 95%, about 50% to about 100%, about 60% to about 70%,about 60% to about 80%, about 60% to about 90%, about 60% to about 95%,about 60% to about 100%, about 70% to about 80%, about 70% to about 90%,about 70% to about 95%, about 70% to about 100%, about 80% to about 90%,about 80% to about 95%, about 80% to about 100%, about 90% to about 95%,about 90% to about 100%, or about 95% to about 100% chemically modifiednucleotides. In some embodiments, the chemically modified tracrRNA maycomprise about 1%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about100% chemically modified nucleotides. In some embodiments, thechemically modified tracrRNA may comprise at least about 1%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or about 95% chemically modified nucleotides. In someembodiments, the chemically modified tracrRNA may comprise at most about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, or about 100% chemically modifiednucleotides.

In some embodiments, the chemically modified tracrRNA may havechemically modified nucleotide on one or more of the positions 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, or 130 in the 5′-3′ direction in thetracrRNA sequence.

In some embodiments, the chemically modified tracrRNA may havechemically modified nucleotide on one or more of the positions 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, or 129 base pairs from the 5′ end of thetracrRNA sequence.

In some embodiments, the chemically modified tracrRNA may havechemically modified nucleotide on one or more of the positions 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, or 129 base pairs from the 3′ end of thetracrRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprisesphosphorothioate (PS) linkages at the 5′ or 3′ end of the gRNA sequence.In some embodiments, the chemically modified gRNA or sgRNA comprises 0,1, 2, 3, 4, or 5 PS linkages at the 5′ end of the gRNA or sgRNAsequence. In some embodiments, the chemically modified gRNA or sgRNAcomprises 0, 1, 2, 3, 4, or 5 PS linkages at the 3′ end of the gRNA orsgRNA sequence. In some embodiments, the chemically modified gRNA orsgRNA comprises 0, 1, 2, 3, 4, or 5 PS linkages, or any combinationsthereof at each of 5′ and 3′ end of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 0, 1, 2 or3 PS linkages, or any combinations thereof at each of 5′ and 3′ end ofthe gRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 1 PS linkage at each of 5′ and 3′ end of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 2 PS linkages at each of 5′ and 3′ end of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 3 PS linkages at each of 5′ and 3′ end of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 4 PS linkages at each of 5′ and 3′ end of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 5 PS linkages at each of 5′ and 3′ end of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 3 PS linkages at the 5′ end and 0 PS linkage(i.e., no modification) at the 3′ end of the gRNA or sgRNA sequence. Insome embodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end and 1 PS linkage at the 3′ end of the gRNA orsgRNA sequence. In some embodiments, the chemically modified gRNA orsgRNA comprises 3 PS linkages at the 5′ end and 2 PS linkages at the 3′end of the gRNA or sgRNA sequence. In some embodiments, the chemicallymodified gRNA or sgRNA comprises 3 PS linkages at the 5′ end and 4 PSlinkages at the 3′ end of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end and 5 PS linkages at the 3′ end of the gRNA orsgRNA sequence. In some embodiments, the chemically modified gRNA orsgRNA comprises 2 PS linkages at the 5′ end and 0 PS linkage (i.e., nomodification) at the 3′ end of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end and 1 PS linkage at the 3′ end of the gRNA orsgRNA sequence. In some embodiments, the chemically modified gRNA orsgRNA comprises 2 PS linkages at the 5′ end and 3 PS linkages at the 3′end of the gRNA or sgRNA sequence. In some embodiments, the chemicallymodified gRNA or sgRNA comprises 2 PS linkages at the 5′ end and 4 PSlinkages at the 3′ end of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end and 5 PS linkages at the 3′ end of the gRNA orsgRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises 2and no more than 2 contiguous phosphorothioate (PS) linkages at the 5′end. In some embodiments, the chemically modified gRNA or sgRNAcomprises 2 and no more than 2 contiguous phosphorothioate (PS) linkagesat the 3′ end. In some embodiments, the chemically modified gRNA orsgRNA comprises 3 contiguous phosphorothioate (PS) linkages at the 5′end. In some embodiments, the chemically modified gRNA or sgRNAcomprises 3 contiguous phosphorothioate (PS) linkages at the 3′ end. Insome embodiments, the chemically modified gRNA or sgRNA comprises thesequence 5′-UsUsU-3′ at the 3′end or at the 5′ end, wherein U indicatesa uridine and wherein s indicates a phosphorothioate (PS) linkage. Insome embodiments, the chemically modified gRNA or sgRNA comprises 3phosphorothioate (PS) linkages at the 5′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 3 phosphorothioate (PS)linkages at the 3′ end. In some embodiments, the chemically modifiedgRNA or sgRNA comprises the two phosphorothioate linkages at the 5′ end,wherein the two phosphorothioate (PS) linkages are two contiguousphosphorothioate (PS) linkages at the first two nucleotide positions ofthe 5′ end. In some embodiments, the chemically modified gRNA or sgRNAcomprises the two phosphorothioate linkages at the 5′ end, wherein thetwo phosphorothioate (PS) linkages are within the first 3-10 nucleotidesof the 5′ end. In some embodiments, the chemically modified gRNA orsgRNA comprises the two phosphorothioate (PS) linkages at the 3′ end,wherein the two phosphorothioate (PS) linkages are two contiguousphosphorothioate (PS) linkages at the first two nucleotide positions ofthe 3′ end. In some embodiments, the chemically modified gRNA or sgRNAcomprises the two phosphorothioate (PS) linkages at the 3′ end, whereinthe two phosphorothioate (PS) linkages are within the first 3-10nucleotides of the 3′ end. In some embodiments, the chemically modifiedgRNA or sgRNA comprises the sequence 5′-UsUsUs-3′ at the 3′ end, whereinU indicates a uridine and s indicates a phosphorothioate (PS) linkage.In some embodiments, the chemically modified gRNA or sgRNA comprises thesequence 5′-UsUsU-3′ at the 3′end, wherein U indicates a uridine and sindicates a phosphorothioate (PS) linkage.

In some embodiments, the chemically modified gRNA or sgRNA comprises PSlinkages at the internal positions of the gRNA or sgRNA sequence. Insome embodiments, the chemically modified gRNA or sgRNA comprises 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 1 PS linkage at the internal position of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 2 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 3 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 4 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 5 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 6 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 7 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 8 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 9 PS linkages at the internal positions of thegRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 10 PS linkages at the internal positions of thegRNA or sgRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises PSlinkages at the 5′ end, the 3′ end, or at the internal positions, or anycombination thereof, of the gRNA or sgRNA sequence. In some embodiments,the chemically modified gRNA or sgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 PS linkages at the 5′end, the 3′ end, or at the internalpositions, or any combination thereof, of the gRNA or sgRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises 2PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 0 PS linkage(i.e., no modification) at the internal position of the gRNA or sgRNAsequence. In some embodiments, the chemically modified gRNA or sgRNAcomprises 2 PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and1 PS linkage at the internal position of the gRNA or sgRNA sequence. Insome embodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end, 3 PS linkages at the 3′ end, and 2 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end, 3 PS linkages at the 3′ end, and 3 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end, 3 PS linkages at the 3′ end, and 4 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end, 3 PS linkages at the 3′ end, and 5 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end, 3 PS linkages at the 3′ end, and 6 PS linkagesat the internal positions of the gRNA or sgRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises 3PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 0 PS linkage(i.e., no modification) at the internal position of the gRNA or sgRNAsequence. In some embodiments, the chemically modified gRNA or sgRNAcomprises 3 PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and1 PS linkage at the internal position of the gRNA or sgRNA sequence. Insome embodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end, 2 PS linkages at the 3′ end, and 2 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end, 2 PS linkages at the 3′ end, and 3 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end, 2 PS linkages at the 3′ end, and 4 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end, 2 PS linkages at the 3′ end, and 5 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end, 2 PS linkages at the 3′ end, and 6 PS linkagesat the internal positions of the gRNA or sgRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises 2PS linkages at the 5′ end, 2 PS linkages at the 3′ end, and 0 PS linkage(i.e., no modification) at the internal position of the gRNA or sgRNAsequence. In some embodiments, the chemically modified gRNA or sgRNAcomprises 2 PS linkages at the 5′ end and 2 PS linkages at the 3′ endand 1 PS linkage at the internal position of the gRNA or sgRNA sequence.In some embodiments, the chemically modified gRNA or sgRNA comprises 2PS linkages at the 5′ end and 2 PS linkages at the 3′ end and 2 PSlinkages at the internal positions of the gRNA or sgRNA sequence. Insome embodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end and 2 PS linkages at the 3′ end and 3 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end and 2 PS linkages at the 3′ end and 4 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end and 2 PS linkages at the 3′ end and 5 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 2 PSlinkages at the 5′ end and 2 PS linkages at the 3′ end and 6 PS linkagesat the internal positions of the gRNA or sgRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises 3PS linkages at the 5′ end, 3 PS linkages at the 3′ end, and 0 PS linkage(i.e., no modification) at the internal position of the gRNA or sgRNAsequence. In some embodiments, the chemically modified gRNA or sgRNAcomprises 3 PS linkages at the 5′ end and 3 PS linkages at the 3′ endand 1 PS linkage at the internal position of the gRNA or sgRNA sequence.In some embodiments, the chemically modified gRNA or sgRNA comprises 3PS linkages at the 5′ end and 3 PS linkages at the 3′ end and 2 PSlinkages at the internal positions of the gRNA or sgRNA sequence. Insome embodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end and 3 PS linkages at the 3′ end and 3 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end and 3 PS linkages at the 3′ end and 4 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end and 3 PS linkages at the 3′ end and 5 PS linkagesat the internal positions of the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA comprises 3 PSlinkages at the 5′ end and 3 PS linkages at the 3′ end and 6 PS linkagesat the internal positions of the gRNA or sgRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprisesadditional modified or unmodified nucleotide (N) with phosphodiesterlinkage, wherein N is an A, C, G, U, dA (deoxyA), dC (deoxyC), dG(deoxyG), or T, and any combinations thereof. In some embodiments, thechemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additionalN with phosphodiester linkage, wherein N is an A, G, U, dA, dG, dC, orT, and any combinations thereof. In some embodiments, the chemicallymodified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additional N withphosphodiester linkage at the 5′ or 3′ end. In one embodiment, thechemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additionalN with phosphodiester linkage at the 5′ end. In a preferred embodiment,the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5additional N with phosphodiester linkage at the 3′ end. In someembodiments, the chemically modified gRNA or sgRNA comprises 1, 2, 3, 4,or 5 additional N with phosphodiester linkage and each N is the samemodified or unmodified nucleotide. For example, the chemically modifiedgRNA or sgRNA may comprise 4 additional N with phosphodiester linkageand each N of the 4 additional N is A, C, G, U, dA, dC, dG, or T. Forexample, the chemically modified gRNA or sgRNA may comprise 4 additionalN with phosphodiester linkage and each N of the 4 additional N is A. Forexample, the chemically modified gRNA or sgRNA may comprise 3 additionalN with phosphodiester linkage and each N of the 3 additional N is A, C,G, U, dA, dC, dG, or T. For example, the chemically modified gRNA orsgRNA may comprise 3 additional N with phosphodiester linkage and each Nof the 3 additional N is A. For example, the chemically modified gRNA orsgRNA may comprise 3 additional N with phosphodiester linkage and each Nof the 3 additional N is C. For example, the chemically modified gRNA orsgRNA may comprise 3 additional N with phosphodiester linkage and each Nof the 3 additional N is G. For example, the chemically modified gRNA orsgRNA may comprise 3 additional N with phosphodiester linkage and each Nof the 3 additional N is U. For example, the chemically modified gRNA orsgRNA may comprise 3 additional N with phosphodiester linkage and each Nof the 3 additional N is dA. For example, the chemically modified gRNAor sgRNA may comprise 3 additional N with phosphodiester linkage andeach N of the 3 additional N is dC. For example, the chemically modifiedgRNA or sgRNA may comprise 3 additional N with phosphodiester linkageand each N of the 3 additional N is dG. For example, the chemicallymodified gRNA or sgRNA may comprise 3 additional N with phosphodiesterlinkage and each N of the 3 additional N is T.

In some embodiments, the chemically modified gRNA or sgRNA comprisesadditional Uracil (U) with phosphodiester linkage at the 5′ or 3′ end.In one embodiment, the chemically modified gRNA or sgRNA comprisesadditional U with phosphodiester linkage at the 5′ end. In a preferredembodiment, the chemically modified gRNA or sgRNA comprises additional Uwith phosphodiester linkage at the 3′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 1, 2, 3, 4, or 5 additionalU with phosphodiester linkage at the 5′ or 3′ end. In some embodiments,the chemically modified gRNA or sgRNA comprises 1 additional U withphosphodiester linkage at the 5′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 1 additional U withphosphodiester linkage at the 3′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 2 additional U withphosphodiester linkage at the 5′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 2 additional U withphosphodiester linkage at the 3′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 3 additional U withphosphodiester linkage at the 5′ end. In a preferred embodiment, thechemically modified gRNA or sgRNA comprises 3 additional U withphosphodiester linkage at the 3′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 4 additional U withphosphodiester linkage at the 5′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 4 additional U withphosphodiester linkage at the 3′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 5 additional U withphosphodiester linkage at the 5′ end. In some embodiments, thechemically modified gRNA or sgRNA comprises 5 additional U withphosphodiester linkage at the 3′ end.

In some embodiments, the chemically modified gRNA or sgRNA comprises aribonebularine (depicted as “X” in this application, e.g., in Table 1 orTable 24). In some embodiments, the chemically modified gRNA or sgRNAcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonebularines. In someembodiments, the chemically modified gRNA or sgRNA does not compriseribonebularine. In some embodiments, the chemically modified gRNA orsgRNA comprises 1 ribonebularine. In some embodiments, the chemicallymodified gRNA or sgRNA comprises 2 ribonebularines. In some embodiments,the chemically modified gRNA or sgRNA comprises 3 ribonebularines. Insome embodiments, the chemically modified gRNA or sgRNA comprises 4ribonebularines. In some embodiments, the chemically modified gRNA orsgRNA comprises 5 ribonebularines. In some embodiments, the chemicallymodified gRNA or sgRNA comprises 6 ribonebularines. In some embodiments,the chemically modified gRNA or sgRNA comprises 7 ribonebularines. Insome embodiments, the chemically modified gRNA or sgRNA comprises 8ribonebularines. In some embodiments, the chemically modified gRNA orsgRNA comprises 9 ribonebularines. In some embodiments, the chemicallymodified gRNA or sgRNA comprises 10 ribonebularines. In someembodiments, the nebularine replaces an adenine in an unmodified gRNA orsgRNA or sgRNA or sgRNA. In some embodiments, the nebularine is in thespacer sequence. In some embodiments, the nebularine is in a tracrRNAsequence. In some embodiments, the nebularine is in a crRNA sequence inthe tracrRNA sequence. In some embodiments, the nebularine is in a stemloop structure in the tracrRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises a2′-O-methylnebularine (depicted as “x” in this application, e.g., inTable 1 or Table 24). In some embodiments, the chemically modified gRNAor sgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 102′-O-methylnebularines. In some embodiments, the chemically modifiedgRNA or sgRNA does not comprise 2′-O-methylnebularine. In someembodiments, the chemically modified gRNA or sgRNA comprises 12′-O-methylnebularine. In some embodiments, the chemically modified gRNAor sgRNA comprises 2 2′-O-methylnebularines. In some embodiments, thechemically modified gRNA or sgRNA comprises 3 2′-O-methylnebularines. Insome embodiments, the chemically modified gRNA or sgRNA comprises 42′-O-methylnebularines. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 5 2′-O-methylnebularines. In some embodiments,the chemically modified gRNA or sgRNA comprises 62′-O-methylnebularines. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 7 2′-O-methylnebularines. In some embodiments,the chemically modified gRNA or sgRNA comprises 82′-O-methylnebularines. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 9 2′-O-methylnebularines. In some embodiments,the chemically modified gRNA or sgRNA comprises 102′-O-methylnebularines. In some embodiments, the 2′-O-methylnebularinereplaces an adenine in an unmodified gRNA or sgRNA or sgRNA or sgRNA. Insome embodiments, the 2′-O-methylnebularine is in the spacer sequence.In some embodiments, the 2′-O-methylnebularine is in a tracrRNAsequence. In some embodiments, the 2′-O-methylnebularine is in a crRNAsequence in the tracrRNA sequence. In some embodiments, the2′-O-methylnebularine is in a stem loop structure in the tracrRNAsequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises a2′-deoxynebularine (depicted as “dX” in this application, e.g., in Table1 or Table 24). In some embodiments, the chemically modified gRNA orsgRNA comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2′-deoxynebularine.In some embodiments, the chemically modified gRNA or sgRNA does notcomprise 2′-deoxynebularine. In some embodiments, the chemicallymodified gRNA or sgRNA comprises 1 2′-deoxynebularine. In someembodiments, the chemically modified gRNA or sgRNA comprises 22′-deoxynebularine. In some embodiments, the chemically modified gRNA orsgRNA comprises 3 2′-deoxynebularines. In some embodiments, thechemically modified gRNA or sgRNA comprises 4 2′-deoxynebularines. Insome embodiments, the chemically modified gRNA or sgRNA comprises 52′-deoxynebularines. In some embodiments, the chemically modified gRNAor sgRNA comprises 6 2′-deoxynebularines. In some embodiments, thechemically modified gRNA or sgRNA comprises 7 2′-deoxynebularines. Insome embodiments, the chemically modified gRNA or sgRNA comprises 82′-deoxynebularines. In some embodiments, the chemically modified gRNAor sgRNA comprises 9 2′-deoxynebularines. In some embodiments, thechemically modified gRNA or sgRNA comprises 10 2′-deoxynebularines. Insome embodiments, the 2′-deoxynebularine replaces an adenine in anunmodified gRNA or sgRNA or sgRNA or sgRNA. In some embodiments, the2′-deoxynebularine is in the spacer sequence. In some embodiments, the2′-deoxynebularine is in a tracrRNA sequence. In some embodiments, the2′-deoxynebularine is in a crRNA sequence in the tracrRNA sequence. Insome embodiments, the 2′-deoxynebularine is in a stem loop structure inthe tracrRNA sequence.

In some embodiments, the chemically modified gRNA or sgRNA comprises a2′-O-methylribonucleotide (2′-OMe). In some embodiments, the chemicallymodified gRNA or sgRNA comprises from about 0 to about 70 2′-OMe. Insome embodiments, the chemically modified gRNA or sgRNA comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 2′-OMe. In someembodiments, the chemically modified gRNA or sgRNAs may comprise fromabout 0 to about 70 2′-OMe. In some embodiments, the chemically modifiedgRNA or sgRNAs may comprise from about 0 to about 10, about 0 to about20, about 0 to about 30, about 0 to about 35, about 0 to about 40, about0 to about 45, about 0 to about 50, about 0 to about 55, about 0 toabout 60, about 0 to about 65, about 0 to about 70, about 10 to about20, about 10 to about 30, about 10 to about 35, about 10 to about 40,about 10 to about 45, about 10 to about 50, about 10 to about 55, about10 to about 60, about 10 to about 65, about 10 to about 70, about 20 toabout 30, about 20 to about 35, about 20 to about 40, about 20 to about45, about 20 to about 50, about 20 to about 55, about 20 to about 60,about 20 to about 65, about 20 to about 70, about 30 to about 35, about30 to about 40, about 30 to about 45, about 30 to about 50, about 30 toabout 55, about 30 to about 60, about 30 to about 65, about 30 to about70, about 35 to about 40, about 35 to about 45, about 35 to about 50,about 35 to about 55, about 35 to about 60, about 35 to about 65, about35 to about 70, about 40 to about 45, about 40 to about 50, about 40 toabout 55, about 40 to about 60, about 40 to about 65, about 40 to about70, about 45 to about 50, about 45 to about 55, about 45 to about 60,about 45 to about 65, about 45 to about 70, about 50 to about 55, about50 to about 60, about 50 to about 65, about 50 to about 70, about 55 toabout 60, about 55 to about 65, about 55 to about 70, about 60 to about65, about 60 to about 70, or about 65 to about 70 2′-OMe. In someembodiments, the chemically modified gRNA or sgRNAs may comprise fromabout 0, about 10, about 20, about 30, about 35, about 40, about 45,about 50, about 55, about 60, about 65, or about 70 2′-OMe. In someembodiments, the chemically modified gRNA or sgRNAs may comprise from atleast about 0, about 10, about 20, about 30, about 35, about 40, about45, about 50, about 55, about 60, or about 65 2′-OMe. In someembodiments, the chemically modified gRNA or sgRNAs may comprise from atmost about 10, about 20, about 30, about 35, about 40, about 45, about50, about 55, about 60, about 65, or about 70 2′-OMe. In someembodiments, the chemically modified gRNA or sgRNA does not comprise2′-OMe. In some embodiments, the chemically modified gRNA or sgRNAcomprises 62 2′-OMe. In some embodiments, the chemically modified gRNAor sgRNA comprises 54 2′-OMe.

In some embodiments, the chemically modified gRNA or sgRNA comprises2′-OMe at the 5′ end of the gRNA or sgRNA sequence. In some embodiments,the chemically modified gRNA or sgRNA comprises 2′-OMe at the 3′ end ofthe gRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 2′-OMe at the internal positions of the gRNA orsgRNA sequence. In some embodiments, the chemically modified gRNA orsgRNA comprises 2′-OMe at the 5′ end, 3′ end, or internal positions ofthe gRNA or sgRNA sequence. In some embodiments, the chemically modifiedgRNA or sgRNA comprises 2′-OMe at the 5′ end, 3′ end, and internalpositions of the gRNA or sgRNA sequence. In some embodiments, thechemically modified gRNA or sgRNA does not comprise 2′-OMe at thepositions 2, 3, 4, 23, 24, 25, 27, 31, or 42, or any combinationsthereof, in the 5′-3′ direction in the gRNA or sgRNA sequence. In someembodiments, the chemically modified gRNA or sgRNA does not comprise2′-OMe at the positions 2, 3, 4, 23, 24, 25, 27, 31, and 42 in the 5′-3′direction in the gRNA or sgRNA sequence.

An exemplary chemically modified gRNA or sgRNA sequence is shown below:

(SEQ ID NO: 55) 5′-csasgsGUUCCAUGGGAUGCUCUgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgca ccgAgUCggugcusususu-3′wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

An exemplary chemically modified spacer sequence is shown below:

(SEQ ID NO: 56) 5′-csasgsGUUCCAUGGGAUGCUCU-3′wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

An exemplary chemically modified tracrRNA sequence is shown below:

(SEQ ID NO: 57) 5′-gUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3′wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified gRNA or sgRNA sequence is shownbelow:

(SEQ ID NO: 55) 5′-csasgsGUUCCAUGGGAUGCUCUgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGca ccgagucggugcusususu-3′wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified tracrRNA sequence is shown below:

(SEQ ID NO: 57) 5′-gUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 58) 5′-cscscsGCACCTTGGCGCAGCGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu-3′,wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 59) 5′-asasgsAUACCUGAAUAACCCUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu-3′(GA091)

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 60) 5′-asasgsAUACCUGAAUAACCCUCGUUUUAGAgcuagaaauagcAAGUUAAAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggca ccgagucggugcusususu3′(GA098)wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 59) 5′-asasgsAUACCUGAAUAACCCUCgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgca ccgAgUCggugcusususu-3′(GAO99)wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 59) 5′- asasgsAUACCUGAAUAACCCUCgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGc accgagucggugcusususu-3′(GA100)wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 11) 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGca ccgagucggugcusususuuuu-3′(GA385)wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 11) 5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGca ccgagucggugcusususuuUu-3′(GA386),wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Another exemplary chemically modified guide RNA sequence is shown below:

(SEQ ID NO: 12) 5′-cscscsGCACCUUGGCGCAGCGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcac cgagucggugcuususuuuu-3′,(GA387)wherein the uppercase A, U, G, or C denote a ribonucleotide adenosine,uridine, guanine, or cytidine, the lowercase a, u, g, and c indicate2′-O-Methyl- modified adenine, uridine, guanine, and cytidine, and “s”denotes phosphorothioate (PS) linkage.

Additional chemically modified guide RNA sequences and target geneinformation are provided in Table 1 or Table 24.

In some embodiments, the chemical modification comprises aphosphorothioate linkage (PS). In some embodiments, the sgRNA comprisesa phosphorothioate linkage (PS) at a 5′ end or at a 3′ end. In someembodiments, the sgRNA comprises two and no more than two contiguousphosphorothioate linkages (PS) at the 5′ end or at the 3′ end. In someembodiments, the sgRNA comprises three contiguous phosphorothioatelinkages (PS) at the 5′ end or at the 3′ end. In some embodiments, thesgRNA comprises the sequence 5′-UsUsU-3′ at the 3′end or at the 5′ end,wherein U indicates a uridine and wherein s indicates a phosphorothioatelinkage (PS).

In some aspects, provided herein, is an sgRNA that comprises (i) aspacer sequence and (ii) a tracrRNA sequence, wherein the spacersequence hybridizes with a target polynucleotide sequence in a PCSK9gene or an ANGPTL3 gene when contacted with the target polynucleotidesequence, wherein the tracrRNA sequence binds a Cas protein in a baseeditor system when contacted with the base editor system, and whereinthe sgRNA comprises a nebularine or a deoxynebularine. In someembodiments, the nebularine or the deoxynebularine replaces an adeninein an unmodified sgRNA. In some embodiments, the nebularine or thedeoxynebularine is in the spacer sequence. In some embodiments, whereinthe nebularine or the deoxynebularine is in the tracrRNA sequence. Insome embodiments, the nebularine or the deoxynebularine is in a tracrRNAsequence. In some embodiments, the nebularine or the deoxynebularine isin a crRNA sequence in the tracrRNA sequence. In some embodiments, thenebularine or the deoxynebularine is in a stem loop structure in thetracrRNA sequence.

In some aspects, provided herein, is a single guide RNA that comprises asequence selected from Table 1 or Table 24, wherein a, u, g, and cindicate 2′-OMe modified adenine, uridine, guanine, and cytidine,wherein s indicates a phosphorothioate (PS) linkage.

In some embodiments, the chemically modified guide RNA comprises anunmodified tracrRNA sequenceGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 61). In some embodiments, thechemically modified guide RNA comprises a tracrRNA sequenceGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 61), which comprises 1 or moremodification.

In some embodiments, the chemically modified guide RNA comprises anoligonucleotide that is at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, atleast 99.7%, or at least 99.9% identical to any one of the chemicallymodified guide RNAs listed in Table 1 or Table 24. In some embodiments,the chemically modified guide RNA comprises any one of the chemicallymodified guide RNAs listed in Table 1 or Table 24. In some embodiments,the chemically modified guide RNA is any one of the chemically modifiedguide RNAs listed in Table 1 or Table 24. In some embodiments, thechemically modified guide RNA comprises an oligonucleotide that is atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.7%, or at least99.9% identical to any one of the chemically modified guide RNAs of SEQID NOs: 9-11, 55, 59, 253-452, 1618-1635, 1637-1800, 1802-2135, and2191. In some embodiments, the chemically modified guide RNA comprisesany one of the chemically modified guide RNAs of SEQ ID NOs: 9-11, 55,59, 253-452, 1618-1635, 1637-1800, 1802-2135, and 2191. In someembodiments, the chemically modified guide RNA is any one of thechemically modified guide RNAs of SEQ ID NOs: 9-11, 55, 59, 253-452,1618-1635, 1637-1800, 1802-2135, and 2191.

Base Editing at Splice Sites

In an aspect, provided herein are base editor systems and methods ofusing same for modifying target genes by generating a genetic alterationin the DNA sequence that occurs at the boundary of an exon and an intron(splice site) at a splice site of a target gene. In some embodiments,the base editor system modifies a nucleobase at a splice acceptor siteof a target gene. In some embodiments, the base editor system modifies anucleobase at a splice donor site of a target gene. Modification at asplice donor or splice acceptor site may result in alternatedtranscripts. In some embodiments, modification at a splice donor orsplice acceptor site results in aberrant transcripts. In someembodiments, modification at a splice donor or splice acceptor siteresults in unstable transcripts that are subject to degradation upontranscription. In some embodiments, modification at a splice donor orsplice acceptor site results in a premature stop codon in thetranscript. In certain embodiments, the methods comprise knocking out orknocking down genes by targeting splice acceptor-splice donor (SA-SD)sites or premature STOP (pmSTOP) sites. For such methods, guidepolynucleotides are designed to disrupt one or more slice acceptor/donorsites within the target nucleotide sequence. In some embodiments, aguide polynucleotide, e.g. a single guide RNA comprises a sequence of atleast 10 contiguous nucleotides or a sequence of 17-23 contiguousnucleotides, that is complementary to a target sequence in the genome ofan organism and comprises a target base pair.

Disruptions at SA-SD sites are particularly advantageous because one mayknock out coding sequence and non-coding RNAs (ncRNAs) without stopcodon read through. Efficiency of base editing can be determined on thegenomic level by EditR analysis of Sanger sequencing traces or by nextgeneration sequencing (NGS), and also on the protein level by flowcytometry. Splice acceptor-splice donor base editing gRNAs that targetthe splice donor regions and the splice acceptor region exhibit baseconversion efficiency of at least 5% and, in some cases, at least 80% orgreater (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%). In some cases, SA-SD gRNAsare significantly more efficient at C-to-T conversions than gRNAs thatintroduce premature stop codons disrupting.

Guide RNAs for targeting SA-SD sites can be designed using an R basedprogram that identifies gRNAs targeting all ncRNAs and protein codinggene SA-SD sites. In some cases, the user supplies the reference genome,Ensembl transcript ID of the reference sequence, protospacer adjacentmotif (PAM) site, and distance to subset upstream and downstream ofexon-intron boundary. The program extracts sequences of 20 basepairs+the PAM length upstream and 15 base pairs downstream of anexon-intron boundary, as well as the splice site motif. In someembodiments, a guide molecule can be from 20 to 120 bases in length, ormore. In certain embodiments, a guide molecule can be from 20 to 60bases in length, or 20 to 50 bases, or 30 to 50 bases, or 39 to 46bases.

In some cases, it is advantageous to use chemically modified gRNAshaving increased stability when transfected into mammalian cells. Forexample, gRNAs can be chemically modified to comprise 2′-O-methylphosphorthioate modifications on at least one 5′ nucleotide and at leastone 3′ nucleotide of each gRNA. In some cases, the three terminal 5′nucleotides and three terminal 3′ nucleotides are chemically modified tocomprise 2′-O-methyl phosphorthioate modifications.

In an aspect, provided herein are base editing systems and methods fortargeting diseases for base editing disruption. The target sequence canbe any disease-associated polynucleotide or gene, as have beenestablished in the art.

Without being bound by a particular theory, where the goal is to disrupta gene in vivo for a therapeutic purpose, cytosine base editors may beused to directly introduce stop codons into the coding sequence of thegene (nonsense mutations) by altering specific codons for glutamine(CAG-TAG, CAA-TAA), arginine (CGA-TGA), and tryptophan (TGG-TAG/TAA/TGA,with editing of cytosines on the antisense strand). In certainembodiments, adenine base editors may be used to disrupt gene functionand/or expression by modifying nucleobases at splice sites of targetgenes. The more favorable off-target profile of adenine base editors,particularly with respect to gRNA-independent off-target DNA baseediting, recommend the use of adenine base editors over cytosine baseeditors for therapeutic purposes. In some embodiments, a base editor,e.g. an adenosine nucleobase editor as describe herein may be used todisrupt a splice donors at the 5′ ends of introns or splice acceptors atthe 3′ ends of introns. In some embodiments, splice site disruptionresults in the inclusion of intronic sequences in messenger RNA(mRNA)—potentially introducing nonsense, frameshift, or in-frame indelmutations that result in premature stop codons or in insertion/deletionof amino acids that disrupt protein activity—or in the exclusion ofexonic sequences, which can also introduce nonsense, frameshift, orin-frame indel mutations.

Canonical splice donors comprise the DNA sequence GT on the sensestrand, whereas canonical splice acceptors comprise the DNA sequence AG.In some embodiments, a base editor, e.g. an adenosine nucleobase editoras described herein can be used to generate alteration of the sequencedisrupts normal splicing. In some embodiments, the adenosine base editordisrupts a complementary base in the second position in the antisensestrand (GT-GC). In some embodiments, the adenosine base editor disruptsthe first position in the sense strand (AG-GG).

Examples of useful applications of mutation or disruption of anendogenous gene sequence that reduces or abolishes expression of thetarget gene related to the disease, with significantly reducedoff-target effect, indels frequency, and/or other unintended geneticinterruptions related to double stranded breaks that result fromtraditional CRISPR-Cas9 nuclease modification. In some embodiments, thetarget gene is a PCSK9 gene. In some embodiments, the target gene is anANGPTL3 gene.

The chemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the binding affinity between a programmable DNAbinding component of a base editor fusion protein, such as a Cas protein(e.g., a Cas9 protein, a nuclease inactive Cas9 protein, a Cas9 nickase)and guide RNAs (e.g., gRNAs or sgRNAs). In some embodiments, thechemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the binding affinity between a Cas protein and gRNAsor sgRNAs by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%,120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%,240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%,900%, or 1000% compared to unmodified gRNAs or sgRNAs. In someembodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can enhance or increase the binding affinity between a Casprotein, e.g. a Cas9 protein, a nuclease inactive Cas9 protein, a Cas9nickase, and gRNAs or sgRNAs by about 20% to about 300%, about 50% toabout 300%, about 100% to about 300%, about 150% to about 300%, about20% to about 50%, about 20% to about 100%, about 20% to about 150%,about 20% to about 200%, about 20% to about 250%, about 50% to about100%, about 50% to about 150%, about 50% to about 200%, about 50% toabout 250%, about 100% to about 150%, about 100% to about 200%, about100% to about 250%, about 150% to about 200%, about 150% to about 250%,about 200% to about 250%, at least about 20%, at least about 50%, atleast about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300% compared to unmodified gRNAs orsgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs ofthe present disclosure can enhance or increase the binding affinitybetween a base editor fusion or Cas protein and gRNAs or sgRNAs by about20% to about 300%, about 50% to about 300%, about 100% to about 300%,about 150% to about 300%, about 20% to about 50%, about 20% to about100%, about 20% to about 150%, about 20% to about 200%, about 20% toabout 250%, about 50% to about 100%, about 50% to about 150%, about 50%to about 200%, about 50% to about 250%, about 100% to about 150%, about100% to about 200%, about 100% to about 250%, about 150% to about 200%,about 150% to about 250%, about 200% to about 250%, at least about 20%,at least about 50%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, or at least about 300% comparedunmodified gRNAs or sgRNAs. In some embodiments, the chemically modifiedgRNAs or sgRNAs of the present disclosure can enhance or increase thebinding affinity between a Cas protein or component of a base editorfusion protein and gRNAs or sgRNAs by at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared tounmodified gRNAs or sgRNAs.

The chemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the binding affinity between a programmable DNAbinding component of a base editor fusion protein, such as a Cas protein(e.g., a Cas9 protein, a nuclease inactive Cas9 protein, a Cas9 nickase)and the tracrRNA sequence of the guide RNAs (e.g., gRNAs or sgRNAs). Insome embodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can enhance or increase the binding affinity between a baseeditor fusion or Cas protein and the tracrRNA sequence of the gRNAs orsgRNAs by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%,130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%,250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%,or 1000% compared to unmodified gRNAs or sgRNAs. In some embodiments,the chemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the binding affinity between a base editor fusion orCas protein and the tracrRNA sequence of the gRNAs or sgRNAs by about20% to about 300%, about 50% to about 300%, about 100% to about 300%,about 150% to about 300%, about 20% to about 50%, about 20% to about100%, about 20% to about 150%, about 20% to about 200%, about 20% toabout 250%, about 50% to about 100%, about 50% to about 150%, about 50%to about 200%, about 50% to about 250%, about 100% to about 150%, about100% to about 200%, about 100% to about 250%, about 150% to about 200%,about 150% to about 250%, about 200% to about 250%, at least about 20%,at least about 50%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, or at least about 300% compared tounmodified gRNAs or sgRNAs. In some embodiments, the chemically modifiedgRNAs or sgRNAs of the present disclosure can enhance or increase thebinding affinity between a base editor fusion or Cas protein and thetracrRNA sequence of the gRNAs or sgRNAs by about 20% to about 300%,about 50% to about 300%, about 100% to about 300%, about 150% to about300%, about 20% to about 50%, about 20% to about 100%, about 20% toabout 150%, about 20% to about 200%, about 20% to about 250%, about 50%to about 100%, about 50% to about 150%, about 50% to about 200%, about50% to about 250%, about 100% to about 150%, about 100% to about 200%,about 100% to about 250%, about 150% to about 200%, about 150% to about250%, about 200% to about 250%, at least about 20%, at least about 50%,at least about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300% compared unmodified gRNAs or sgRNAs.In some embodiments, the chemically modified gRNAs or sgRNAs of thepresent disclosure can enhance or increase the binding affinity betweena base editor fusion or Cas protein and the tracrRNA sequence of thegRNAs or sgRNAs by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold,3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs orsgRNAs.

In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs maybind a programmable DNA binding component of a base editor fusionprotein, such as a Cas protein (,) with increased binding affinitycompared to a tracrRNA sequence in an unmodified sgRNA. The type II Casprotein may be used as a system or base editor as described herein. Insome embodiments, the tracrRNA sequence of the gRNAs or sgRNAs may binda Type II Cas protein with at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%,220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%,700%, 800%, 900%, or 1000% increased binding affinity compared to atracrRNA sequence in an unmodified sgRNA. In some embodiments, thetracrRNA sequence of the gRNAs or sgRNAs may bind a base editorcomprising a Type II Cas protein with at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold increased bindingaffinity compared to a tracrRNA sequence in an unmodified sgRNA. In someembodiments, the tracrRNA sequence of the gRNAs or sgRNAs may bind abase editor fusion protein comprising a Cas9 protein with increasedbinding affinity compared to a tracrRNA sequence in an unmodified sgRNA.In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs maybind a Cas9 protein with at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%,220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, or 300% increasedbinding affinity compared to a tracrRNA sequence in an unmodified sgRNA.In some embodiments, the tracrRNA sequence of the gRNAs or sgRNAs maybind a base editor fusion protein comprising a Cas9 protein with atleast 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, ormore than 10-fold increased binding affinity compared to a tracrRNAsequence in an unmodified sgRNA.

The chemically modified gRNAs or sgRNAs of the present disclosure canreduce off-target effect in gene modification (e.g., gene or genomeediting) of the target sequence. In some embodiments, the chemicallymodified gRNAs or sgRNAs of the present disclosure can reduce off-targeteffect in gene modification of the target sequence by at least 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%,35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%,300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodifiedgRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs orsgRNAs of the present disclosure can reduce off-target effect in genemodification of the target sequence by about 20% to about 300%, about50% to about 300%, about 100% to about 300%, about 150% to about 300%,about 20% to about 50%, about 20% to about 100%, about 20% to about150%, about 20% to about 200%, about 20% to about 250%, about 50% toabout 100%, about 50% to about 150%, about 50% to about 200%, about 50%to about 250%, about 100% to about 150%, about 100% to about 200%, about100% to about 250%, about 150% to about 200%, about 150% to about 250%,about 200% to about 250%, at least about 20%, at least about 50%, atleast about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300% compared to unmodified gRNAs orsgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs ofthe present disclosure can reduce off-target effect in gene modificationof the target sequence by about 20% to about 300%, about 50% to about300%, about 100% to about 300%, about 150% to about 300%, about 20% toabout 50%, about 20% to about 100%, about 20% to about 150%, about 20%to about 200%, about 20% to about 250%, about 50% to about 100%, about50% to about 150%, about 50% to about 200%, about 50% to about 250%,about 100% to about 150%, about 100% to about 200%, about 100% to about250%, about 150% to about 200%, about 150% to about 250%, about 200% toabout 250%, at least about 20%, at least about 50%, at least about 100%,at least about 150%, at least about 200%, at least about 250%, or atleast about 300% compared unmodified gRNAs or sgRNAs. In someembodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can reduce off-target effect in gene modification of thetarget sequence by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold,3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or more than 10-fold compared to unmodified gRNAs orsgRNAs.

In some embodiments, the target polynucleotide sequence is in a genome,wherein the gRNA or sgRNA is capable of directing a component (e.g.Cas9) of a base editor fusion protein to effect a modification in thetarget polynucleotide sequence, and wherein the modification results inless off-target effect in the genome as compared to an unmodified gRNAor sgRNA. In some embodiments, the gRNA or sgRNA is capable of directinga Type II Cas protein to effect a modification in the targetpolynucleotide sequence wherein the modification results in at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%,290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% less off-targeteffect in the genome as compared to an unmodified gRNA or sgRNA. In someembodiments, the gRNA or sgRNA is capable of directing a Type II Casprotein of a base editor fusion protein to effect a modification in thetarget polynucleotide sequence wherein the modification results in atleast 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, ormore than 10-fold less off-target effect in the genome as compared to anunmodified gRNA or sgRNA. In some embodiments, the target polynucleotidesequence is in a genome, wherein the gRNA or sgRNA is capable ofdirecting a Cas9 protein to effect a modification in the targetpolynucleotide sequence, and wherein the modification results in lessoff-target effect in the genome as compared to an unmodified gRNA orsgRNA. In some embodiments, the gRNA or sgRNA is capable of directing aCas9 protein to effect a modification in the target polynucleotidesequence wherein the modification results in at least 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%,40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%,300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% less off-target effectin the genome as compared to an unmodified gRNA or sgRNA. In someembodiments, the gRNA or sgRNA is capable of directing a Cas9. In someembodiments, the target polynucleotide sequence is in a genome, whereinthe gRNA or sgRNA is capable of directing a base editor fusion proteinto effect a modification in the target polynucleotide sequence, andwherein the modification results in less off-target effect in the genomeas compared to an unmodified gRNA or sgRNA. In some embodiments, thegRNA or sgRNA is capable of directing a base editor fusion protein toeffect a modification in the target polynucleotide sequence wherein themodification results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%,220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%,700%, 800%, 900%, or 1000% less off-target effect in the genome ascompared to an unmodified gRNA or sgRNA. In some embodiments, the gRNAor sgRNA is capable of directing a base editor fusion protein to effecta modification in the target polynucleotide sequence wherein themodification results in at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold,3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or more than 10-fold less off-target effect in thegenome as compared to an unmodified gRNA or sgRNA. In some embodiments,the modification using the chemically modified gRNAs or sgRNAs resultsin less off-target effect in a cell as compared to an unmodified gRNA orsgRNA. In some embodiments, the modification using the chemicallymodified gRNAs or sgRNAs results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%,600%, 700%, 800%, 900%, or 1000% less off-target effect in a cell ascompared to an unmodified gRNA or sgRNA. In some embodiments, themodification using the chemically modified gRNAs or sgRNAs results in atleast 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, ormore than 10-fold less off-target effect in a cell as compared to anunmodified gRNA or sgRNA.

The chemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the gene modification (e.g., gene or genome editing)efficiency. In some embodiments, the chemically modified gRNAs or sgRNAsof the present disclosure can enhance or increase the gene modification(e.g., gene or genome editing) efficiency by at least 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%,40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%,300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000% compared to unmodifiedgRNAs or sgRNAs. In some embodiments, the chemically modified gRNAs orsgRNAs of the present disclosure can enhance or increase the genemodification (e.g., gene or genome editing) efficiency by about 20% toabout 300%, about 50% to about 300%, about 100% to about 300%, about150% to about 300%, about 20% to about 50%, about 20% to about 100%,about 20% to about 150%, about 20% to about 200%, about 20% to about250%, about 50% to about 100%, about 50% to about 150%, about 50% toabout 200%, about 50% to about 250%, about 100% to about 150%, about100% to about 200%, about 100% to about 250%, about 150% to about 200%,about 150% to about 250%, about 200% to about 250%, at least about 20%,at least about 50%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, or at least about 300% compared tounmodified gRNAs or sgRNAs. In some embodiments, the chemically modifiedgRNAs or sgRNAs of the present disclosure can enhance or increase thegene modification (e.g., gene or genome editing) efficiency by about 20%to about 300%, about 50% to about 300%, about 100% to about 300%, about150% to about 300%, about 20% to about 50%, about 20% to about 100%,about 20% to about 150%, about 20% to about 200%, about 20% to about250%, about 50% to about 100%, about 50% to about 150%, about 50% toabout 200%, about 50% to about 250%, about 100% to about 150%, about100% to about 200%, about 100% to about 250%, about 150% to about 200%,about 150% to about 250%, about 200% to about 250%, at least about 20%,at least about 50%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, or at least about 300% comparedunmodified gRNAs or sgRNAs. In some embodiments, the chemically modifiedgRNAs or sgRNAs of the present disclosure can enhance or increase thegene modification (e.g., gene or genome editing) efficiency by at least1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than10-fold compared to unmodified gRNAs or sgRNAs.

The chemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the stability of the complex of a base editorprotein or component (e.g., Cas9 protein) and the guide RNAs (e.g.,gRNAs or sgRNAs), for example, the stability of base editor complex. Insome embodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can enhance or increase the stability of the Cas protein-gRNAcomplex by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, %, 11%, 12%,13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%,120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%,240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%,900%, or 1000% compared to unmodified gRNAs or sgRNAs. In someembodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can enhance or increase the stability of the base editorfusion or Cas protein-gRNA complex by about 20% to about 300%, about 50%to about 300%, about 100% to about 300%, about 150% to about 300%, about20% to about 50%, about 20% to about 100%, about 20% to about 150%,about 20% to about 200%, about 20% to about 250%, about 50% to about100%, about 50% to about 150%, about 50% to about 200%, about 50% toabout 250%, about 100% to about 150%, about 100% to about 200%, about100% to about 250%, about 150% to about 200%, about 150% to about 250%,about 200% to about 250%, at least about 20%, at least about 50%, atleast about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300% compared to unmodified gRNAs orsgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs ofthe present disclosure can enhance or increase the stability of the baseeditor fusion or Cas protein-gRNA complex by about 20% to about 300%,about 50% to about 300%, about 100% to about 300%, about 150% to about300%, about 20% to about 50%, about 20% to about 100%, about 20% toabout 150%, about 20% to about 200%, about 20% to about 250%, about 50%to about 100%, about 50% to about 150%, about 50% to about 200%, about50% to about 250%, about 100% to about 150%, about 100% to about 200%,about 100% to about 250%, about 150% to about 200%, about 150% to about250%, about 200% to about 250%, at least about 20%, at least about 50%,at least about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300% compared unmodified gRNAs or sgRNAs.In some embodiments, the chemically modified gRNAs or sgRNAs of thepresent disclosure can enhance or increase the stability of the baseeditor fusion or Cas protein-gRNA complex by at least 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold comparedto unmodified gRNAs or sgRNAs.

In some embodiments, the chemically modified gRNAs or sgRNAs of thepresent disclosure can enhance or increase the stability of the baseeditor complex by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%,120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%,240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%,900%, or 1000% compared to unmodified sgRNAs. In some embodiments, thechemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the stability of the base editor complex by about20% to about 300%, about 50% to about 300%, about 100% to about 300%,about 150% to about 300%, about 20% to about 50%, about 20% to about100%, about 20% to about 150%, about 20% to about 200%, about 20% toabout 250%, about 50% to about 100%, about 50% to about 150%, about 50%to about 200%, about 50% to about 250%, about 100% to about 150%, about100% to about 200%, about 100% to about 250%, about 150% to about 200%,about 150% to about 250%, about 200% to about 250%, at least about 20%,at least about 50%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, or at least about 300% compared tounmodified sgRNAs. In some embodiments, the chemically modified gRNAs orsgRNAs of the present disclosure can enhance or increase the stabilityof the base editor complex by about 20% to about 300%, about 50% toabout 300%, about 100% to about 300%, about 150% to about 300%, about20% to about 50%, about 20% to about 100%, about 20% to about 150%,about 20% to about 200%, about 20% to about 250%, about 50% to about100%, about 50% to about 150%, about 50% to about 200%, about 50% toabout 250%, about 100% to about 150%, about 100% to about 200%, about100% to about 250%, about 150% to about 200%, about 150% to about 250%,about 200% to about 250%, at least about 20%, at least about 50%, atleast about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300% compared unmodified sgRNAs. In someembodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can enhance or increase the stability of the baseeditor-sgRNA or Cas9-sgRNA complex by at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold compared tounmodified sgRNAs.

The chemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the stability of the guide RNAs (e.g., gRNAs orsgRNAs) in vitro, in vivo, and/or ex vivo. In some embodiments, thechemically modified gRNAs or sgRNAs of the present disclosure canenhance or increase the stability of the gRNAs or sgRNAs in vitro, invivo, and/or ex vivo by at least 1%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9% 10%,11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%,110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%,230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%,800%, 900%, or 1000% compared to unmodified gRNAs or sgRNAs. In someembodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can enhance or increase the stability of the gRNAs or sgRNAsin vitro, in vivo, and/or ex vivo by about 20% to about 300%, about 50%to about 300%, about 100% to about 300%, about 150% to about 300%, about20% to about 50%, about 20% to about 100%, about 20% to about 150%,about 20% to about 200%, about 20% to about 250%, about 50% to about100%, about 50% to about 150%, about 50% to about 200%, about 50% toabout 250%, about 100% to about 150%, about 100% to about 200%, about100% to about 250%, about 150% to about 200%, about 150% to about 250%,about 200% to about 250%, at least about 20%, at least about 50%, atleast about 100%, at least about 150%, at least about 200%, at leastabout 250%, or at least about 300% compared to unmodified gRNAs orsgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs ofthe present disclosure can enhance or increase the stability of thegRNAs or sgRNAs in vitro, in vivo, and/or ex vivo by about 20% to about300%, about 50% to about 300%, about 100% to about 300%, about 150% toabout 300%, about 20% to about 50%, about 20% to about 100%, about 20%to about 150%, about 20% to about 200%, about 20% to about 250%, about50% to about 100%, about 50% to about 150%, about 50% to about 200%,about 50% to about 250%, about 100% to about 150%, about 100% to about200%, about 100% to about 250%, about 150% to about 200%, about 150% toabout 250%, about 200% to about 250%, at least about 20%, at least about50%, at least about 100%, at least about 150%, at least about 200%, atleast about 250%, or at least about 300% compared unmodified gRNAs orsgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAs ofthe present disclosure can enhance or increase the stability of thegRNAs or sgRNAs in vitro, in vivo, and/or ex vivo by at least 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold comparedto unmodified gRNAs or sgRNAs.

In some embodiments, the chemically modified gRNAs or sgRNAs exhibitincreased stability in a cell as compared to unmodified gRNAs or sgRNAs.In some embodiments, the chemically modified gRNAs or sgRNAs exhibit atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%,270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, or 1000%higher or increased stability in a cell as compared to unmodified gRNAsor sgRNAs. In some embodiments, the chemically modified gRNAs or sgRNAsexhibit be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, or more than 10-fold higher or increased stability in a cell ascompared to unmodified gRNAs or sgRNAs.

In some embodiments, the stability of the chemically modified gRNAs orsgRNAs is measured by half-life of the gRNAs or sgRNAs in the cell. Insome embodiments, the chemically modified gRNAs or sgRNAs exhibitincreased half-life in the cell compared to unmodified gRNAs or sgRNAs.In some embodiments, the gRNAs or sgRNAs exhibit increased half-life inthe cell by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%,130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%,250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%,or 1000% as compared to unmodified gRNAs or sgRNAs. In some embodiments,the gRNAs or sgRNAs exhibit increased half-life in the cell by about 20%to about 300%, about 50% to about 300%, about 100% to about 300%, about150% to about 300%, about 20% to about 50%, about 20% to about 100%,about 20% to about 150%, about 20% to about 200%, about 20% to about250%, about 50% to about 100%, about 50% to about 150%, about 50% toabout 200%, about 50% to about 250%, about 100% to about 150%, about100% to about 200%, about 100% to about 250%, about 150% to about 200%,about 150% to about 250%, about 200% to about 250%, at least about 20%,at least about 50%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, or at least about 300% as compared tounmodified gRNAs or sgRNAs. In some embodiments, the gRNAs or sgRNAsexhibit increased half-life in the cell by at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold as compared tounmodified gRNAs or sgRNAs.

The chemically modified gRNAs or sgRNAs of the present disclosure can bemore resistant to degradation as compared to an unmodified gRNA orsgRNA. In some embodiments, the chemically modified gRNAs or sgRNAs ofthe present disclosure can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%,100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%,220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%, 600%,700%, 800%, 900%, or 1000% more resistant to degradation compared tounmodified gRNAs or sgRNAs. In some embodiments, the chemically modifiedgRNAs or sgRNAs of the present disclosure can be about 20% to about300%, about 50% to about 300%, about 100% to about 300%, about 150% toabout 300%, about 20% to about 50%, about 20% to about 100%, about 20%to about 150%, about 20% to about 200%, about 20% to about 250%, about50% to about 100%, about 50% to about 150%, about 50% to about 200%,about 50% to about 250%, about 100% to about 150%, about 100% to about200%, about 100% to about 250%, about 150% to about 200%, about 150% toabout 250%, about 200% to about 250%, at least about 20%, at least about50%, at least about 100%, at least about 150%, at least about 200%, atleast about 250%, or at least about 300% more resistant to degradationcompared to unmodified gRNAs or sgRNAs. In some embodiments, thechemically modified gRNAs or sgRNAs of the present disclosure can beabout 20% to about 300%, about 50% to about 300%, about 100% to about300%, about 150% to about 300%, about 20% to about 50%, about 20% toabout 100%, about 20% to about 150%, about 20% to about 200%, about 20%to about 250%, about 50% to about 100%, about 50% to about 150%, about50% to about 200%, about 50% to about 250%, about 100% to about 150%,about 100% to about 200%, about 100% to about 250%, about 150% to about200%, about 150% to about 250%, about 200% to about 250%, at least about20%, at least about 50%, at least about 100%, at least about 150%, atleast about 200%, at least about 250%, or at least about 300% moreresistant to degradation compared unmodified gRNAs or sgRNAs. In someembodiments, the chemically modified gRNAs or sgRNAs of the presentdisclosure can be at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold,3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or more than 10-fold more resistant to degradationcompared to unmodified gRNAs or sgRNAs.

Target Gene Modification

In some aspects, provided herein, is a method for treating or preventinga condition in a subject in need thereof, the method comprisingadministering to the subject the composition or lipid nanoparticlecomprising chemically modified gRNAs or sgRNAs described herein, whereinthe gRNAs or sgRNAs direct a base editor protein to effect amodification in a target polynucleotide sequence in a cell of thesubject, thereby treating or preventing the condition. In someembodiments, the target polynucleotide is in a PCSK9 gene. In someembodiments, the target polynucleotide is in an ANGPTL3 gene. In someembodiments, the modification is at a splice site of the targetpolynucleotide. In some embodiments, the modification is at a splicedonor site of the target polynucleotide. In some embodiments, themodification is at a splice acceptor site of the target polynucleotide.In some embodiments, the modification reduces expression of functionalPCSK9 protein encoded by the PCSK9 gene in the subject. In someembodiments, the modification reduces expression of functional ANGPTL3protein encoded by the ANGPTL3 gene in the subject. In some embodiments,the condition is atherosclerotic vascular disease. In some embodiments,the condition is an atherosclerotic vascular disease,hypertriglyceridemia, or diabetes. In some embodiments, the subjectexhibits a reduced blood LDL cholesterol level and/or a reduced bloodtriglycerides level as compared to before the administration.

In some embodiments, the disclosure provides base-editing systems,compositions and methods for editing a polynucleotide encoding anApolipoprotein C3 (APOC3) protein and variants thereof. In someembodiments, provided herein are genome/base-editing systems,compositions and methods for editing a polynucleotide encodingProprotein convertase subtilisin/kexin type 9 (PCSK9) and variantsthereof. In some embodiments, provided herein are genome/base-editingsystems, compositions and methods for editing a polynucleotide encodingAngiopoietin-like 3 (ANGPTL3) and variants thereof. To edit a targetgene, a target gene polynucleotide may contact the compositionsdisclosed herein comprising a sgRNA and a adenosine base editor protein,wherein the sgRNA comprises a spacer sequence and a tracrRNA sequence,wherein the spacer sequence hybridizes with a target polynucleotidesequence in a PCSK9 or ANGPTL3 gene and the tracrRNA sequence binds theadenosine base editor protein (e.g. a Cas9 component of the adenosinebase editor). In some embodiments, the sgRNA therefore directs the baseeditor protein to the target polynucleotide sequence to result in a to Gmodification in the target gene. In some embodiments, the target gene ortarget polynucleotide is selected from a gene encoding PCSK9, APOC3,LPA, and ANGPTL3. In some embodiments, the target polynucleotidesequence is in a PCSK9 gene. In some embodiments, the targetpolynucleotide sequence is in an ANGPTL3 gene. In some embodiments, themodification reduces or abolishes expression of functional PCSK9 proteinencoded by the PCSK9 gene in the cell. In some embodiments, themodification reduces or abolishes expression of functional ANGPTL3protein encoded by the ANGPTL3 gene in the cell. In some embodiments,the introduction is performed via a lipid nanoparticle that comprisesthe composition.

For example, the sgRNA and the Adenosine base editor protein may beexpressed in a cell where a target gene editing is desired (e.g., aliver cell), to thereby allowing contact of the target gene with thecomposition disclosed herein (e.g., sgRNA and the Adenosine base editorprotein). In some embodiments, the binding of the Adenosine base editorprotein to its target polynucleotide sequence in the target gene isdirected by a single guide RNA disclosed herein, e.g., a single guideRNA comprising (i) a spacer sequence and (ii) a tracrRNA sequence,wherein the spacer sequence hybridizes with a target polynucleotidesequence in a target gene. Thus, by designing the guide RNA sequence,the Adenosine base editor protein can be directed to edit any targetpolynucleotide sequence in the target gene (e.g., target gene encodingPCSK9, APOC3 and ANGPTL3). In some embodiments, the guide RNA sequenceis co-expressed with the Adenosine base editor protein in a cell whereediting is desired.

In some embodiments, a target gene comprising more than one mutationsdescribed herein are contemplated. For example, a target gene encoding avariant protein can be produced using the methods described herein thatincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. To makemultiple mutations in the target gene, a plurality of guide RNAsequences can be used, each guide RNA sequence targeting one targetpolynucleotide sequence in the target gene. The Adenosine base editorprotein is capable of editing each and every target polynucleotidesequence dictated by the guide RNA sequence. For example, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more guide RNA sequences can be used in a geneediting reaction. In some embodiments, the guide RNA sequences as used(e.g., gRNA). In some embodiments, DNA molecule encoding the guide RNAsequences can also be used.

In some embodiments, simultaneous modifications into more than onetarget genes (e.g., more than one target gene in the LDL-mediatedcholesterol clearance pathway) are also contemplated herein. Forexample, in some embodiments, a modification may be simultaneouslyintroduced into PCSK9 and APOC3 gene. In some embodiments, amodification may be simultaneously introduced into PCSK9 and LDL-R gene.In some embodiments, a modification may be simultaneously introducedinto PCSK9 and IODL gene. In some embodiments, a modification may besimultaneously introduced into PCSK9 and LPA gene. In some embodiments,a modification may be simultaneously introduced into APOC3 and IODLgene. In some embodiments, a modification may be simultaneouslyintroduced into LDL-R and APOC3 gene. In some embodiments, amodification may be simultaneously introduced into LDL-R and IDOL gene.In some embodiments, a modification may be simultaneously introducedinto PCSK9, APOC3, LDL-R and IDOL gene. In some embodiments, amodification can be simultaneously introduced into PCSK9 and ANGPTL3gene. To simultaneously introduce of modifications into more than onetarget genes, multiple guide nucleotide sequences are used.

To edit a gene encoding the PCSK9 or ANGPTL3 protein, the gene iscontacted with the composition described herein. In some embodiments, atarget polynucleotide sequence in a target gene is contacted with thesingle guide RNA disclosed herein and a Adenosine base editor protein ora nucleic acid sequence encoding the Adenosine base editor protein,wherein the single guide RNA directs the Adenosine base editor proteinto effect a modification in the target gene (e.g., target gene encodingPCSK9, APOC3 and ANGPTL3). In some embodiments, the targetpolynucleotide sequence is the gene locus in the genomic DNA of a cell.In some embodiments, the cell is a cultured cell. In some embodiments,the cell is in vivo. In some embodiments, the cell is in vitro. In someembodiments, the cell is ex vivo.

In some embodiments, the cell is from a mammal. In some embodiments, themammal is a human. In some embodiments, the mammal is a rodent. In someembodiments, the rodent is a mouse. In some embodiments, the rodent is arat. As would be understood be those skilled in the art, a targetpolynucleotide sequence may be a DNA molecule comprising a coding strandand a complementary strand, e.g., the PCSK9 or ANGPTL3 gene locus in agenome. As such, the target polynucleotide sequence may also includecoding regions (e.g., exons) and non-coding regions (e.g., introns orsplicing sites). In some embodiments, the target polynucleotide sequenceis located in the coding region (e.g., an exon) of the target gene(e.g., the PCSK9 or ANGPTL3 gene locus). As such, the modification inthe coding region may result in an amino acid change in the proteinencoded by the target gene, i.e., a mutation. In some embodiments, themutation is a loss of function mutation. In some embodiments, theloss-of-function mutation is a naturally occurring loss-of-functionmutation. In some embodiments, the target polynucleotide sequence islocated in a non-coding region of the target gene, e.g., in an intron ora splicing site. In some embodiments, the base editor system providedherein results in a A•T to G•C alteration at a splice site in a PCSK9gene or a ANGPTL3 gene. In some embodiments, the A•T to G•C alterationis at a splice acceptor site of the PCSK9 gene. In some embodiments, theA•T to G•C alteration results in an aberrant PCSK9 transcript encoded bythe PCSK9 gene. In some embodiments, the A•T to G•C alteration resultsin a non-functional PCSK9 polypeptide encoded by the PCSK9 gene whenexpressed in a cell. In some embodiments, the A•T to G•C alteration isat a 5′ end of a splice donor site of an intron 1 of the PCSK9 gene. Insome embodiments, the A•T to G•C alteration is at a 5′ end of a splicedonor site of an intron 4 of the PCSK9 gene. In some embodiments, thebase editor system provided herein results in a A•T to G•C alteration ata splice site ANGPTL3 gene, or the second A•T to G•C alteration is at asplice donor site of the ANGPTL3 gene. In some embodiments, the A•T toG•C alteration or the second A•T to G•C alteration is at a spliceacceptor site of the ANTPTL3 gene. In some embodiments, the A•T to G•Calteration or the second A•T to G•C alteration results in an aberrantANGPTL3 transcript encoded by the ANGPTL3 gene. In some embodiments, theA•T to G•C alteration or the second A•T to G•C alteration results in anon-functional ANGPTL3 polypeptide encoded by the ANGPTL3 gene. In someembodiments, the A•T to G•C alteration or the second A•T to G•Calteration is at a 5′ end of a splice donor site of an intron 6 of theANGPLT3 gene.

In some embodiments, a target polynucleotide sequence is located in asplicing site and the editing of such sequence causes alternativesplicing of the mRNA of a target gene. In some embodiments, thealternative splicing leads to leading to loss-of-function mutants. Insome embodiments, the alternative splicing leads to the introduction ofa premature stop codon in a mRNA encoded by the target gene, resultingin truncated and unstable proteins. In some embodiments, mutants thatare defective in folding are produced. A loss-of-function variantgenerated by a gene that is modified using the compositions and methodsdisclosed herein, may have reduced activity compared to a wild typeprotein encoded by an unmodified target gene. Activity refers to anyknown biological activity of the wild-type protein in the art.

In some embodiments, the activity of a loss-of-function variant may bereduced by at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 99%, ormore. In some embodiments, the loss-of-function variant has no more than50%, no more than 40%, no more than 30%, no more than 20%, no more than10%, no more than 5%, no more than 1% or less activity compared to awild type protein.

In some embodiments, cellular activity of a protein encoded by a targetgene may be reduced by reducing the level of properly folded and activeprotein. Introducing destabilizing mutations into the wild type proteinmay cause misfolding or deactivation of the protein. A variant generatedby modifying a target gene using the compositions and methods disclosedherein comprises one or more destabilizing mutations may have reducedactivity compared to the wild type protein encoded by an unmodifiedtarget gene. For example, the activity of a variant comprising one ormore destabilizing mutations may be reduced by at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 99%, or more.

In some embodiments, the methods and composition disclosed hereinreduces or abolishes expression and/or function of protein encoded by atarget gene. For example, the methods and composition disclosed hereinreduces expression and/or function of protein encoded by the target geneby at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold,at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-foldrelative to a control.

In some embodiments, the methods and composition disclosed hereinreduces or abolishes expression and/or function of the protein encodedby a target gene by at least 2-fold relative to a control. For example,the methods and composition disclosed herein reduces or abolishesexpression and/or function of the protein encoded by a target gene by atleast 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, or at least 10-foldrelative to a control.

Some aspects of the present disclosure provide strategies of editingtarget gene to reduce the amount of full-length, functional proteinbeing produced. In some embodiments, stop codons may be introduced intothe coding sequence of target gene upstream of the normal stop codon(referred to as a “premature stop codon”). Premature stop codons causepremature translation termination, in turn resulting in truncated andnonfunctional proteins and induces rapid degradation of the mRNA via thenon-sense mediated mRNA decay pathway. See, e.g., Baker et al., CurrentOpinion in Cell Biology 16 (3): 293-299, 2004; Chang et al, AnnualReview of Biochemistry 76: 51-74, 2007; and Behm-Ansmant et ah, Genes &Development 20 (4): 391-398, 2006, each of which is incorporated hereinby reference.

Proprotein Convertase Subtilisin-Kexin Type 9 (PCSK9)

In some embodiments, the target gene for modification using thecompositions and methods disclosed herein is gene encoding PCSK9.Proprotein convertase subtilisin-kexin type 9 (PCSK9), also known asneural apoptosis-regulated convertase 1 (NARC-I), is a proteinase K-likesubtilase identified as the 9th member of the secretory subtilasefamily. “Proprotein convertase subtilisin/kexin type 9 (PCSK9)” refersto an enzyme encoded by the PCSK9 gene. PCSK9 binds to the receptor forlow-density lipoprotein (LDL) particles. In the liver, the LDL receptorremoves LDL particles from the blood through the endocytosis pathway.When PCSK9 binds to the LDL receptor, the receptor is channeled towardsthe lysosomal pathway and broken down by proteolytic enzymes, limitingthe number of times that a given LDL receptor is able to uptake LDLparticles from the blood. Thus, blocking PCSK9 activity may lead to moreLDL receptors being recycled and present on the surface of the livercells, and will remove more LDL cholesterol from the blood.

Therefore, blocking PCSK9 can lower blood cholesterol levels. PCSK9orthologs are found across many species. PCSK9 is inactive when firstsynthesized, a pre-pro enzyme, because a section of the peptide chainblocks its activity; proprotein convertases remove that section toactivate the enzyme. Pro-PCSK9 is a secreted, globular, serine proteasecapable of proteolytic auto-processing of its N-terminal pro-domain intoa potent endogenous inhibitor of PCSK9, which blocks its catalytic site.PCSK9's role in cholesterol homeostasis has been exploited medically.Drugs that block PCSK9 can lower the blood level of low-densitylipoprotein cholesterol (LDL-C). The first two PCSK9 inhibitors,alirocumab and evolocumab, were approved by the U.S. Food and DrugAdministration in 2015 for lowering cholesterol where statins and otherdrugs were insufficient.

The human gene for PCSK9 localizes to human chromosome Ip33-p34.3. PCSK9is expressed in cells capable of proliferation and differentiationincluding, for example, hepatocytes, kidney mesenchymal cells,intestinal ileum, and colon epithelia as well as embryonic braintelencephalon neurons. See, e.g., Seidah et al., 2003 PNAS 100:928-933,which is incorporated herein by reference.

Original synthesis of PCSK9 is in the form of an inactive enzymeprecursor, or zymogen, of 72-kDa, which undergoes autocatalytic,intramolecular processing in the endoplasmic reticulum (ER) to activateits functionality. This internal processing event has been reported tooccur at the SSVFAQ jSIP motif, and has been reported as a requirementof exit from the ER. “j” indicates cleavage site. See, Benjannet et al.,2004 J. Biol. Chem. 279:48865-48875, and Seidah et al, 2003 PNAS100:928-933, each of which are incorporated herein by reference. Thecleaved protein is then secreted. The cleaved peptide remains associatedwith the activated and secreted enzyme.

The gene sequence for human PCSK9 is ˜22-kb long with 12 exons encodinga 692 amino acid protein. The protein sequence of human PCSK9 can befound, for example, at Deposit No. NP_777596.2, which sequence isincorporated herein in its entirety. Human, mouse and rat PCSK9 nucleicacid sequences have been deposited; see, e.g., GenBank Accession Nos.:AX127530 (also AX207686), AX207688, and AX207690, respectively, each ofwhich sequence is incorporated herein in its entirety. The gene sequenceof Macaca fascicularis can be found publically, for example, NCBI GeneID: 102142788, which sequence is incorporated herein in their entirety.Macaca fascicularis proprotein convertase subtilisin/kexin type 9isoform X2 sequence can be found publically, for example, at NCBIReference Sequence: XP_005543317.1, which sequence is incorporatedherein in its entirety.

The translated protein contains a signal peptide in the NH2-terminus,and in cells and tissues an about 74 kDa zymogen (precursor) form of thefull-length protein is found in the endoplasmic reticulum. Duringinitial processing in the cell, the about 14 kDa prodomain peptide isautocatalytically cleaved to yield a mature about 60 kDa proteincontaining the catalytic domain and a C-terminal domain often referredto as the cysteine-histidine rich domain (CHRD). This about 60 kDa formof PCSK9 is secreted from liver cells. The secreted form of PCSK9appears to be the physiologically active species, although anintracellular functional role of the about 60 kDa form has not beenruled out.

Numerous PCSK9 variants are disclosed and/or claimed in several patentpublications including, but not limited to the following: PCTPublication Nos. WO2001031007, WO2001057081, WO2002014358, WO2001098468,WO2002102993, WO2002102994, WO2002046383, WO2002090526, WO2001077137,and WO2001034768; US Publication Nos. US 2004/0009553 and US2003/0119038, and European Publication Nos. EP 1 440 981, EP 1 067 182,and EP 1 471 152, each of which are incorporated herein by reference.

Several mutant forms of PCSK9 are well characterized, including S 127R,N157K, F216L, R218S, and D374Y, with S 127R, F216L, and D374Y beinglinked to autosomal dominant hypercholesterolemia (ADH). Benjannet etal. (J. Biol. Chem., 279(47):48865-48875 (2004)) demonstrated that the S127R and D374Y mutations result in a significant decrease in the levelof pro-PCSK9 processed in the ER to form the active secreted zymogen. Asa consequence, it is believed that wild-type PCSK9 increases theturnover rate of the LDL receptor causing inhibition of LDL clearance(Maxwell et al, PNAS, 102(6):2069-2074 (2005); Benjannet et al, andLalanne et al), while PCSK9 autosomal dominant mutations result inincreased levels of LDLR, increased clearance of circulating LDL, and acorresponding decrease in plasma cholesterol levels. See, Rashid et al,PNAS, 102(15):5374-5379 (2005); Abifadel et al, 2003 Nature Genetics 34:154-156; Timms et al, 2004 Hum. Genet. 114:349-353; and Leren, 2004Clin. Genet. 65:419-422, each of which are incorporated herein byreference.

A later-published study on the S127R mutation of Abifadel et al,reported that patients carrying such a mutation exhibited higher totalcholesterol and apoB 100 in the plasma attributed to (1) anoverproduction of apoB 100-containing lipoproteins, such as low densitylipoprotein (LDL), very low density lipoprotein (VLDL) and intermediatedensity lipoprotein (IDL), and (2) an associated reduction in clearanceor conversion of said lipoproteins. Together, the studies referencedabove evidence the fact that PCSK9 plays a role in the regulation of LDLproduction. Expression or upregulation of PCSK9 is associated withincreased plasma levels of LDL cholesterol, and inhibition or the lackof expression of PCSK9 is associated with low LDL cholesterol plasmalevels. Significantly, lower levels of LDL cholesterol associated withsequence variations in PCSK9 have conferred protection against coronaryheart disease; Cohen et al, 2006 N. Engl. J. Med. 354: 1264-1272.

Lalanne et al. demonstrated that LDL catabolism was impaired andapolipoprotein B-containing lipoprotein synthesis was enhanced in twopatients harboring S 127R mutations in PCSK9 (J. Lipid Research, 46:1312-1319 (2005)). Sun et al. also provided evidence that mutant formsof PCSK9 are also the cause of unusually severe dominanthypercholesterolaemia as a consequence of its effect of increasingapolipoprotein B secretion (Sun et al, Hum. Mol. Genet, 14(9): 1161-1169(2005)). These results were consistent with earlier results whichdemonstrated adenovirus-mediated overexpression of PCSK9 in mice resultsin severe hypercholesteromia due to drastic decreases in the amount ofLDL receptor Dubuc et al., Thromb. Vase. Biol., 24: 1454-1459 (2004), inaddition to results demonstrating mutant forms of PCSK9 also reduce thelevel of LDL receptor (Park et al., J. Biol. Chem., 279:50630-50638(2004). The overexpression of PCSK9 in cell lines, includingliver-derived cells, and in livers of mice in vivo, results in apronounced reduction in LDLR protein levels and LDLR functional activitywithout changes in LDLR mRNA level (Maxwell et al., Proc. Nat. Amer.Set, 101:7100-7105 (2004); Benjannet S. et al, J. Bio. Chem. 279:48865-48875 (2004)).

Various therapeutic approaches to the inhibition of PSCK9 have beenproposed, including: inhibition of PSCK9 synthesis by gene silencingagents, e.g., RNAi; inhibition of PCSK9 binding to LDLR by monoclonalantibodies, small peptides or adnectins; and inhibition of PCSK9autocatalytic processing by small molecule inhibitors. These strategieshave been described in Hedrick et al., Curr Opin Investig Drugs 2009;10:938-46; Hooper et al, Expert Opin Biol Ther, 2013; 13:429-35; Rhaindset al, Clin Lipid, 2012; 7:621-40; Seidah et al, Expert Opin TherTargets 2009; 13:19-28; and Seidah et al, Nat Rev Drug Discov 2012;11:367-83, each of which are incorporated herein by reference.

In some embodiments, the loss of function mutation induced in PCSK9e.g., G106R, L253F, A443T, R93C, etc. In some embodiments, theloss-of-function mutation is engineered (i.e., not naturally occurring),e.g., G24D, S47F, R46H, S 153N, H193Y, etc.

PCSK9 variants that can be useful in the present disclosure areloss-of-function variants that may boost LDL receptor-mediated clearanceof LDL cholesterol, alone or in combination with other genes involved inthe pathway, e.g., APOC3, LDL-R, or Idol. In some embodiments, the PCSK9loss-of-function variants produced using the methods of the presentdisclosure express efficiently in a cell. In some embodiments, the PCKS9loss-of-function variants produced using the methods of the presentdisclosure is activated and exported to engage the clathrin-coated pitsfrom unmodified cells in a paracrine mechanism, thus competing with thewild-type PCSK9 protein. In some embodiments, the PCSK9 loss-of-functionvariant comprises mutations in residues in the LDL-R bonding region thatmake direct contact with the LDL-R protein. In some embodiments, theresidues in the LDL-R bonding region that make direct contact with theLDL-R protein are selected from the group consisting of R194, R237,F379, S372, D374, D375, D378, R46, R237, and A443.

As described herein, a loss-of-function PCSK9 variant, may have reducedactivity compared to a wild type PCSK9 protein. PCSK9 activity refers toany known biological activity of the PCSK9 protein in the art. Forexample, in some embodiments, PCSK9 activity refers to its proteaseactivity. In some embodiments, PCSK9 activity refers to its ability tobe secreted through the cellular secretory pathway. In some embodiments,PCSK9 activity refers to its ability to act as a protein-binding adaptorin clathrin-coated vesicles. In some embodiments, PCSK9 activity refersto its ability to interact with LDL receptor. In some embodiments, PCSK9activity refers to its ability to prevent LDL receptor recycling. Theseexamples are not meant to be limiting.

In some embodiments, the activity of a loss-of-function PCSK9 variantmay be reduced by at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least99%, or more. In some embodiments, the loss-of-function PCSK9 varianthas no more than 50%, no more than 40%, no more than 30%, no more than20%, no more than 10%, no more than 5%, no more than 1% or less activitycompared to a wild type PCSK9 protein. Non-limiting, exemplary assaysfor determining PCSK9 activity have been described in the art, e.g., inUS Patent Application Publication US20120082680, which are incorporatedherein by reference.

In some embodiments, cellular PCSK9 activity may be reduced by reducingthe level of properly folded and active PCSK9 protein. Introducingdestabilizing mutations into the wild type PCSK9 protein may causemisfolding or deactivation of the protein. A PCSK9 variant comprisingone or more destabilizing mutations described herein may have reducedactivity compared to the wild type PCSK9 protein. For example, theactivity of a PCSK9 variant comprising one or more destabilizingmutations described herein may be reduced by at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 99%, or more.

In some embodiments, the methods and composition disclosed herein reduceor abolish expression of protein encoded by a target gene and/orfunction thereof. For example, the methods and composition disclosedherein reduces expression and/or function of PCSK9 protein encoded bythe PCSK9 gene by at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold relative to a control. For example, the methods andcomposition disclosed herein reduces expression and/or function of APOC3protein encoded by the APOC3 gene by at least 3-fold, at least 4-fold,at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold relative to a control. For example,the methods and composition disclosed herein reduces expression and/orfunction of ANGPTL3 protein encoded by the ANGPTL3 gene by at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, or at least 10-fold relativeto a control.

In some embodiments, the gene modification methods and compositionsdisclosed herein reduces expression of functional PCSK9 protein encodedby the PCSK9 gene in the cell by at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 98%, at least 99%, at least99.5%, at least 99.9%, or 100%. In some embodiments, the modificationreduces expression of functional PCSK9 protein encoded by the PCSK9 genein the cell by at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, atleast 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, atleast 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, atleast 200 fold, at least 300 fold, at least 400 fold, at least 500 fold,at least 600 fold, at least 700 fold, at least 800 fold, at least 900fold, at least 1000 fold, at least 2000 fold, at least 3000 fold, atleast 4000 fold, at least 5000 fold, at least 6000 fold, at least 7000fold, at least 8000 fold, at least 9000 fold, or at least 10000 fold. Insome embodiments, the modification abolishes expression of functionalPCSK9 protein encoded by the PCSK9 gene in the cell.

Some aspects of the present disclosure provide strategies of reducingcellular PCSK9 activity via preventing PCSK9 mRNA maturation andproduction. In some embodiments, such strategies involve alterations ofsplicing sites in the PCSK9 gene. Altered splicing site may lead toaltered splicing and maturation of the PCSK9 mRNA. For example, in someembodiments, an altered splicing site may lead to the skipping of anexon, in turn leading to a truncated protein product or an alteredreading frame. In some embodiments, an altered splicing site may lead totranslation of an intron sequence and premature translation terminationwhen an in frame stop codon is encountered by the translating ribosomein the intron. In some embodiments, a start codon is edited and proteintranslation initiates at the next ATG codon, which may not be in thecorrect coding frame.

The splicing sites typically comprises an intron donor site, a Lariatbranch point, and an intron acceptor site. The mechanism of splicing isfamiliar to those skilled in the art. As a non-limiting example, theintron donor site has a consensus sequence of GGGTRAGT, and the C basespaired with the G bases in the intron donor site consensus sequence maybe targeted by the methods and compositions described herein, therebyaltering the intron donor site. The Lariat branch point also hasconsensus sequences, e.g., YTRAC, wherein Y is a pyrimidine and R is apurine. The C base in the Lariat branch point consensus sequence may betargeted by the nucleobase editors described herein, leading to theskipping of the following exon. The intron acceptor site has a consensussequence of YNCAGG, wherein Y is a pyrimidine and N is any nucleotide.The C base of the consensus sequence of the intron acceptor site, andthe C base paired with the G bases in the consensus sequence of theintron acceptor site may be targeted by the nucleobase editors describedherein, thereby altering the intron acceptor site, in turn leading theskipping of an exon. As described herein, gene sequence for human PCSK9is −22-kb long and contains 12 exons and 11 introns. Each of theexon-intron junction may be altered to disrupt the processing andmaturation of the PCSK9 mRNA.

In some embodiments, a splice site disruption generated by a base editorsystem disclosed herein can result in the inclusion of intronicsequences in messenger RNA (mRNA) encoded by the PCSK9 gene. In someembodiments, the splice site disruption generates a nonsense,frameshift, or an in-frame indel mutation that result in premature stopcodons or in insertion/deletion of amino acids that disrupt proteinactivity. In some embodiments, the splice site disruption generatesexclusion of exonic sequences. In some embodiments, the splice sitedisruption generates exclusion of exonic sequences that results innonsense, frameshift, or in-frame indel mutations in the PCSK9transcript. Canonical splice donors comprise the DNA sequence GT on thesense strand, whereas canonical splice acceptors comprise the DNAsequence AG. Alteration of the sequence disrupts normal splicing. Splicedonors can be disrupted by adenine base editing of the complementarybase in the second position in the antisense strand (GT to GC), andsplice acceptors can be disrupted by adenine base editing of the firstposition in the sense strand (AG to GG).

Further, the present disclosure also contemplates the use ofdestabilizing mutations to counteract the effect of gain-of-functionPCSK9 variant. Gain-of-function PCSK9 variants (e.g., thegain-of-function variants have been described in the art and are foundto be associated with hypercholesterolemia (e.g., in Peterson et al., JLipid Res. 2008 June; 49(6): 1152-1156; Benjannet et al., J Biol Chem.2012 Sep. 28;287(40):33745-55; Abifadel et al, Atherosclerosis. 2012August; 223(2):394-400; and Cameron et al, Hum. Mol. Genet. (1 May 2006)15(9): 1551-1558, each of which is incorporated herein by reference).Introducing destabilizing mutations into these gain-of-function PCSK9variants may cause misfolding and deactivation of these gain-of-functionvariants, thereby counteracting the hyper-activity caused by thegain-of-function mutation. Further, gain-of-function mutations inseveral other key factors in the LDL-R mediated cholesterol clearancepathway, e.g., LDL-R, APOB, or APOC, have also been described in theart. Thus, making destabilizing mutations in these factors to counteractthe deleterious effect of the gain-of-function mutation using thecompositions and methods described herein, is also within the scope ofthe present disclosure. As such, the present disclosure further providesmutations that cause misfolding of PCSK9 protein or structurallydestabilization of PCSK9 protein.

The polypeptide and coding nucleic acid sequences of PCSK9 and of othermembers of the family of human origin and those of a number of animalsare publicly available, e.g., from the NCBI website or ENSEMBL website.Examples include, but are not limited to the following sequences, eachof which sequences are incorporated herein in their entireties;

Wild Type PCSK9 Gene (NG 009061.1), Homo sapiens proprotein convertasesubtilisin/kexin type 9 (PCSK9), RefSeqGene (LRG 275) on chromosome 1(SEQ ID NO: 5) GTCCGATGGGGCTCTGGTGGCGTGATCTGCGCGCCCCAGGCGTCAAGCACCCACACCCTAGAAGGTTTCCGCAGCGACGTCGAGGCGCTCATGGTTGCAGGCGGGCGCCGCCGTTCAGTTCAGGGTCTGAGCCTGGAGGAGTGAGCCAGGCAGTGAGACTGGCTCGGGCGGGCCGGGACGCGTCGTTGCAGCAGCGGCTCCCAGCTCCCAGCCAGGATTCCGCGCGCCCCTTCACGCGCCCTGCTCCTGAACTTCAGCTCCTGCACAGTCCTCCCCACCGCAAGGCTCAAGGCGCCGCCGGCGTGGACCGCGCACGGCCTCTAGGTCTCCTCGCCAGGACAGCAACCTCTCCCCTGGCCCTCATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTGCGCCAAGGTGCGGGTGTAGGGATGGGAGGCCGGGGCGAACCCGCAGCCGGGACGGTGCGGTGCTGTTTCCTCTCGGGCCTCAGTTTCCCCCCATGTAAGAGAGGAAGTGGAGTGCAGGTCGCCGAGGGCTCTTCGCTTGGCACGATCTTGGGGACTGCAGGCAAGGCGGCGGGGGAGGACGGGTAGTGGGGAGCACGGTGGAGAGCGGGGACGGCCGGCTCTTTGGGGACTTGCTGGGGCGTGCGGCTGCGCTATTCAGTGGGAAGGTTCGCGGGGTTGGGAGACCCGGAGGCCGAGGAAGGGCGAGCAGAGCACTGCCAGGATATCCTGCCCAGATTTCCCAGTTTCTGCCTCGCCGCGGCACAGGTGGGTGAAGGAGTGAATGCCTGGAACGTACTGGGAACTGCACCAGGCACAGAGAAAGCGGGCTTGCCATTATAGTGGGTTCCGATTTGGTTTGGAAAACATGGGCAGCGGAGGGTGGAGGGCCTGGAGAGAAGGCCCTACCCGAGACAGGGGCGGGGTGGGAAGGACGGCAGATGCTGGGAGCACGAGGCAATTTCTTTATGACACAGAACTCATGCTCTAGTATTCCATCTGTTTCAGCCGAAGAAAAGAACCAGCTGAAGGGGCAGGGGAGAAGGGGCGGAGGTATTCTCGAGGCCCATTGGCGTCCTTTAGGACTCAGGCAGGGAAGGGCCCTTGGTGCTCTGGAGCCGGAGGTGGTGCGCCTGGTACTGGGACCCCGGAGCTGAGCCCGGCGCCTCAGCCCACCTGGCTGTCTGCCGACCGTGTGCGGGGCGAGTTTGCTCAACAACTCTGCCAGCTTCTGGCCCTCAGGCTGTGGGAAGCTTCTTCCCGGGGCGAGACCACTAGCTTTTTCTAAGTATTACCAGCCCAGGACTTGGCTGAGGTTCTGTGTCCCCCAGCTTGGAGTCAGATGTGGGGTTGAATCTTGGCTTCCTCTCACTAGCTGTGGTGCTTGACAAGTCACTTATCCTTGAGCCTCCATTGCCTAATCTTTAAAAGGGAGGTGACAATCGTCCCTACGGCTCAGTGGCAGCAGATGGGGAGATGAAGGGAAAGTTCTGTTGACCATGAGTGAACTTACAATGCAAGCCCCGGGGGGATCACTTGCAGTTTTGTCCCTGTCTGCAGTGTGACCTGTTGGTGACATTGTCTTTGCTCCAAACCACAGCTCCTGGGGCAGAGGGGAAAATTCTGCCACTCACAGCTGCCTGCCCACGCTTCTGTCTGAGTGTGCTGGGTGGCAGGATGGCAAGTCCTTACTCAGCTCAGTATAGCCCTCTTCCTTGTTCCCTGAGCCTTTGACTTTCTCGAGGGATGTTGTGGGGTTGTGGCCAGGATAAGAAAGGGCATTTCAAGTTACCACTGCTCCAAAACAACTGTTCTGGAAATAGTGAGTACCCCATCCTGAGAGGTGAGTAAGCAGAGGCTGTATGACCACCTGAACCAAGCCCTTGAGGATGTTTCTTCTCTGGTGGAAGTTTGGAACAGGAGCCTCCTCAAGTTCATTTATTCATTCATTCAATGGTTATTTTGTGGGAATCGAATTTAGAATGAAAATATTTTTTGGCAAGCAGAAAATAATTTTTAGACCAATCCTTTTCTTTTAGTCATGAGAAACTGAGGCCCAGAGAGAGGAGGTCACCCCAGGTGCATTAGAACTGGGTTTCCAGAACTGACACTCCACTGCACAGAGTACTCTCCCAATTCATTCAATTTTTATTTAGCGGAAGGCATTTTCAGATGGGTCTTTGAAGCATTAGTAGGAGTTCAGCGATGATGGTGTCATGAGAATTTTATTCTAGGATTAGGAGGTACCATGAACAAAGATACAGAGCTGGGAAAACCAGAGGTGGAAGATAAGGAGCACATGTCCACAGTTCTTTTTCTTTTTTTTTTGAGATGGAGTTTCGCTCTTGTTGCCCAGGCTGGAGTGCAATGGTGCAGTCTCAGCTCACTGCAACATCTGTCTCCCGGGTTCAAGTGGTTCTCCTGCCTCAGCCTCCCAAGAAGCTGGGATTACAGGTACCTGCCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGAAGGGGTTTCACCACGTTGGCCAGGCTAGTCGCAAACTCCTGACCTCCTCAGTGGATCCGAGGAGGTGATCCTCCCGCCTCAGCCTCCCAAAGTGCTCGAATTACAGGTGTGAGCCACCACGCCTGGCCTCCACAGTTCTTTATCCACCGTCTGAAATGTAAAATGTTACGAAAACCAAAAGTTTTTTTTGTGATTTATTTGATGGTAGCACCTGACGTGAACTGACATGAGATTATTTTTAATTTAGTTGTGTGAATATGCATATTCATATATTTTGCTGCATAGATTACAGTATGCAGCTCCAGATTCTTCCAAGCAGACTCTGATTGCCCATTACTGCCTTTCTAAAATCCAAACAAGTTCTGAGGTTCAAAACCGTTTTGGCCCTAAGGCTTTGGGTAAAGGGGGTGGACTCTGTTCTACTCTGACTGGAGTCCAAGATGCATATATACAGAGATATGGGTGATGGGGCTGCAAGGTAGGTTGAGGTAGGGGCCAAGGAGGAGCATGGAGTTTGGACTTGATTCATGAGGCTGTGGGGAGCCAGTGAAGGTTCTTAAGCAGGTATGTCTGCCTGAGAGCAGTTGGAGCAGACAAGAGCTAAAAACCAAACAAATCACCATAGATAGTGGCTGCTATAATTTGTTTGTCCCCTCCAAATCTCATGTGGAAATTTGGTCCTCAGTGTTGGAAGTGGGGCCTAATGGGAGGTGTTTGGGTCATGGGGGAGGAACCCCTGTGAAAGGCTTGGTGCCGTCCTTGTGATAATGAGTAAGTTCTCCCGCTATGATTTCCCTTGAAGGCTGATTATTAAAAAGAGCTTGGCACCTCCCTCTCTTCTCTCTTGCTTCTTCTCTTGCCATGTGATTGATCTCTGCACATGTAGGCTCCCCTTCACCTTCTGCCATCAGTGAAAGCAGCTTAAGGCCCTCACCAGAAGCAGATGCTGGTGCCATGCTTCCTGGAGAGCTTGCAGAATCATGAGCTGAATAAATCCCTTTTCCTTGTAAATTACTCACCTTCAGGTATTCCTTTATATAGCAACACAAAAGGACTAAGACAGTGGCCTTGACTTTTCTCTCTCTTTAAGAAGTGTTGCCTTTGCTCACTTAGTCATCCCTTCTGCCTGCATTTGTAGAGCATCTGGATGGGAGATTTATATAACCGTCACTCTTGACTTTCCCAGCAGGCCTATGTCATAGGTACTGTGGTCTCTACAATACAGCAGAGGTATCTGAGGCTCCGAGAGGTTGAGTGACTTGCTCATGGCTGCACAACCAGTAAATATTGGAGCTGGAATTCAGGTCCACGGTTTCCTGGCTCCAAAGCCCATGATTTTTTCCCTCAATTTATTCTGACTGGGGCATGGGGGAGGGGGTGGCCTTTGGGCAGGGCCACCAGGAGCGACCAGGCCCGTAGAGAGCTGGGTGCAGGTACAGAGGAAAACCTGTTGTCGAGTGTGGCCCGTAGTTCCCATTTTTGCCTGAATGGCACATTTGAAAGTGTTATATAACCATGTGAATAATAATAGTTGGCCTATATGAGTTCTTTAATTTGCTTTTTGGTCCGCATTTGGTAACTTCTTTATCATCTACTATACTCTGTTGTGTCTCTTTTGTTGTAATTTGTAAGTAGGGGTGAGATAAAGTACACCTAGGGTTTGCTGGGTTTCTTCCATGTCATCATGTTCCTCCTTGCATGGGGCCAGGATCCGTGGAGGTTGCCTGGCACCTACGTGGTGGTGCTGAAGGAGGAGACCCACCTCTCGCAGTCAGAGCGCACTGCCCGCCGCCTGCAGGCCCAGGCTGCCCGCCGGGGATACCTCACCAAGATCCTGCATGTCTTCCATGGCCTTCTTCCTGGCTTCCTGGTGAAGATGAGTGGCGACCTGCTGGAGCTGGTGAGCCACCCTTTTTGGGAATGGCACTTCCTGATAGGGCTGGGCCACTGCATATACACTGGGGACTGTGCTTAGTAGGCCCATTGCTGAAAATCAGAAGGGGACAGCAAGTATGTATTGAGCACTTATCGGGTACCAAGCACAGTAACTACTGGCTTTCTGTATAGAATTCCCTTTAAGCCTGGCCATGCCCCAGTGGTACGTCTATCTTCATTTGAAAGACGAGGAGACTGAAGTTCAGAGGGGACCACACAGACAGCTAGGGGTAGAGCCTGGATCAAACCCATTGGTCTGCCTGCCAGCCATTCTTGTGCCAATGCATCTGCTGCCTACGGAAACCTGTAGGGACAAGGCCCTGGGATGTTCAGTGGAGCCTGAGTCATTTTATAAAAAAGCATGACTCTAGGGTCCAAAATTCCTTTGAAGCTGTTGCTATCCAGAGTGAAGTCCCTTCTTTAGGACAGGGTGGCCCTCCTCCCTCCTGGATGTCACATCTTCGGTGGAGGGGCAGAAAGGGGACTGGGTATTCTCCTCACCCTGGCCCTAGTGCTTCAAATCTTAAAAAAACGTTTTTATTTGTGCTTCTGCACCACCTTCTAGCCCACCTCGTTTCCTGGCCTCTAACTTGATGAGAGCGTGTGTCATTTTCACACTGATTCTCCACATGGCAGGCGGTGCTTCTTAGCCTCCTGCAGACAGTGAGGCCCCACGGTCTTGTCCAAGGTCACACAGCGTGTAATGGGCAGGGTCAGAGTCTGGAGTCTGGACCTGGGTCTCCTAGCTGCACTGCACTGCTGCCCCATGGGTTAATCAGCTCAGCATACCGTGGCTGAACAGCTACCTCATACCAAGGCCTGTGGCGCCATGACAGGGATTGACAGGGTCCCTGCCTTGGAAACCCGTAGTCTAAGTAGAGGAGACTGACAAGTCAATGCCTTCCATCAGTCTGCTCAACACACGTTTACCAAGTGCCTACTGTGTGCTGCAGAGGCGAAGATGACACAGCTCAGGCCTTTCCCTTGAGCTTACAGTTCAGGAGGAGAGACTGACCAGTGACTGCCAGTACAGTTGACTATGGGACAATGTGCTCAGCCTTGGGGAGAGACGAAGAAGGTACCCGTATAGCACCAGATGACAGGCACGAGCCCCACAGGCCAGGGCAGCTGCTCAGAGGAGAGTAGGCCAAGCAGAAGGCAAACAGAAGGCTGCAGGCATTTGCCATCGAGAGCTGGACTTCAAACTGGGCATCATACCAGCCTGGGTTCGAGTCCTGCCCAGCCCCTTATTGGCTGTCTAACCCTGAGCAAATCCCTTCACCTCTCTGAGCCTCATTCCTCTATCTGTAAACCAGTTATAATAATTGGAACATTCATTTAAGGACTAAATGAGGTCGTGAAGCATTCAGCAGATGCTAGGTACGGAAACTCGCTGAAGTGGGGGCAGGTTAAGAAGCCTCTGGGGATACGAAGGCATCCAGGGACTAGTTGTGGCAGGAGGCTGTTACCACTTAGGTCTGAAGGGTAAGGAGAGGGAATAGCTTTCCCTCTGCCCAGTTGGAGCCGGTGGCATGGAGGAGAGGCTGCCTGTGGGGAATCACCCGAGGGTTCACCGCTGCCATGCGCAGGGAGTCAGGAGGTAGGGAGGGAGTGGGGCAGATGCACACCATTTTTTTTTTTTTTTGAGACTCTGTTGCCCAGACTGGAGTGCAGTGGTGCCATATCTGCACCTCTGCCTCCCGGGTTCAAGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGTGTGCCACCATGCCTGGCTAATTTTTGTATTTTTAATAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCTCGACCTCAGGTGATCCCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGTCACCGCTCCCAGCTGCTGATGCACTCTTGTCCTTCTAACTCCTGCTAGTGCCTCCCATTGGCTGAGCCCAACTGGAAGCTTTGCAAGGGAGCTGGTGCTGCAGTTTGCACTGAGCAGGCTGGAGAAGGCTGGAGAATAGACTAGGGGACAAACCGAATTGCCAGTGCTGTTATGTCATGATTTAGGCATGGAGTCCAGGGCCTGAGCTTCACTCCATGTCCATCCTGCCCAGAGCCTTGGCACAGCCTGGCTCCCAGACAAGATGTCAAGTTCAGAATCCTTCCTAAAAGGAATCCTCTATGCCAGACCGTGTTGCAGGGATATGGGAGTGCTGGGCTCCCAGCCTGATCAAGGAGCGAGAAAACTCAGGCTCCTAGTCTGTCCTCCGGGGCACTAGCAGGGACAAGGTGGGAGGCTGCTGGGCTGGGATGTGGGGACAGGTTTGATCAGGTAAGGCCAGGCTGTGGCTGTGTTTGCTGCTGTCCAAATGGCTTAAGCAGAGTCCCCCGGCCTCTCTGGCTTCTGCAGGCCTTGAAGTTGCCCCATGTCGACTACATCGAGGAGGACTCCTCTGTCTTTGCCCAGAGCATCCCGTGGAACCTGGAGCGGATTACCCCTCCACGGTACCGGGCGGATGAATACCAGCCCCCCGGTAAGACCCCCATCTGTGCCCTGCCCCACCCCATCTGAGCTGAATCCATTTGCTCTGCCCTGGCCTGGCCTCCCTGCTGGTGGTTTCCACTTCTCGGGGGGCTTTGGGACTCAGCACCTCCACTGACCCCTTTTTTTCTGTCCCATCCCCATCCCCTGCAGCCCCCACTGCCTGCCTTCCTGTTGCCCCACAAATGCAAAAGTCTTGCCTTAAATGATCCTCTTTTCCTTCTTTTCTCTTGTTTTCCTTTTCTCACCATTTGGAATGGCCCAGCAGGCTGCACTTACCTTGGAAGGAGGGTTCATCTGATGGTGACTCTACCTAGGGCCCCCAGGCCTCTATAACTCCCAGTGCCCTGCAGACTGGACCAGATCCTTTAATGGGATAGACACAACCCTGTCTGGGATGCCTCTGCCTACCTTCCTGTTTTGCTGCTCCACCTGCCTCCAGCTCCGTTTGGCTTCCTGGGGCTCCCTGCCTGGGCCACTTTGTGTCTTCCCTCTAGGCCTTTCTTTCCACTGTTCCCTCTGCCTGGTGTGGCCTGGCTATGGAAGGGAGGGAGGAGGAGCGGCCATGGAAAACGGTCTGCATTCTAGCAGGGACTTGCAGGTGGCAATTCAGTCGGGGAAGACTCTAGATGCACCTGGCCTGAGGAGAGAATGAAGGGTTCTAGTTGGACTGTGTTAAGTTTGAGGTGCCCATGGTGTGAGGTCTGGAGCTCAGCGCAGAGATGATGCAATGTGGTGGGTCCATGCAACATGGTGCCAGGACGCAGAGCTTGGGGTGAACTCAGCTTTCACCCCTTACCGGTTCTCGTGGGATCTTGGGAAGCCACTTTCTTCTATGAGCTTTGTCGTTCTTGTCTGTAAAATGGGCACATAACCCTGTCCCTGTCCTTCTCACAGGTTGCTGTGAGACTCCAATGAGTTGAAGGATGTGCAGATGCTTTTGGAAGTGAAAAGTTGGGGGGCTACTGTGTGACTTTGCATACACCCAAACTGTGTGACCTTGCATATGTCTGAGTTGCTGCCATTGCAACAGATCAGAGCTGGTGGGCTGGGTGTGGAGAAAGGGTTTGTGTGGGGGACATCCTCTGGCAAGGGTGGCAGCAGCAGAAGTGAGGGGCCTGGTCGGTCATGTGTGCTGACCCGGCCTGGGCAGCCTGTGGCCAGGGAGAGGACAGCTCCTCTGTAGGAAGAGCCTGTTCCTTTCCAACCAGGTGAGACCTCTTCAGTGGAGCCCTGGAGCCCCCTGTACTCCACATCAGTGCCTCAGGGACCTCCCGGAGCAGGCTAATATCAGAGACCAAGAGGGACACTGGCAGAGGATCACAGAGACCCCAGTCCAGGCAGGGACTGAGAAGATCTTGCCCCCTAAGTTAGTTTCCTAGCACTGCTGTGACAAATTACCACCCCCTCGGTTGGAACAAGTTGATTCTCTGCAGTCCTGGAGGCCAGAAGCCTGAATCAGTGTCGGCAGGACCACTTTCTCCCGGGGGGCTCCAGGGAGAAGCTTCTCTTGCCTCTTCCGTGTCCCAACAGCGGCAGCACACCAATCCCAGCCTCTGTCTTCACACAGCCTTCTCTGTGTCTCTCTCCTCTTCATTGTCTCATAAGGACACTTGTCATTGGATTTAGGGCCCACTGGATCCTCCAGGATGATCTCATGTGGGGAACCTTAACCACATCTGCAAGGACCCTTTTTCCAAATAAGGTCACAGCCACAGGTTGTGGGGGTTAGGATGTGAGTGTATCTCTTTGGCAGCCACTGTTCCCTCCTCTCCCTTGGGCCAGAAGCAGACGTGGGGCCCTTTCTTCCCCATAGGATGCCCATGGATTGCCCCCCTTCCCGCTTCCCCCGAGTGTCTGTGGGAGGTGGCAGGAATGGCAGGCAGGGGTGTGGAACCCCTTCTGGAGTCATATCAAGGGCTTGGCTGGAGGAAGTCCTCCTGGAGCTGTTGGGCTGGCATGGGGCAGGCTGGCTGGGCCCAGCAGCAGCTTCTTCATTCATGGGGAGGCCACAAGCATGGGCCCTAGAGCTGGCTGCCGCCCTCAAACCCAGACCCTGCACTCTTAACTGTGTGACCTTGCATACGTCACTCACCCTCTCTGATCTTCAGGTTCCTCTGCAAAAGGGAGGTAATGATAACCCTCACTCTGGGGGGCTGTTTGGAGGGTTAAATCAGTTATTGCTGTAGCATGCATTTCTCTGTCAGGTATTGAGTGAGGTGCTGTGATTTTAGCCCTGCATTTTTCTTTTCTTACCATTCAATAATAACGTTTTGAGCACCCACTGTGCGCCAGGCACCATATTAGGTGCTGGGGATACAAATGTGAATGAAATGAATGTGGTCTCTTCCCCCAACAGTGTATCCAGAAGATTAATCCATTCCTTAAACAAATGCTACTTGACACAGATTAGTTCTGGATAGGCTGAGAGCTCTGAAGGAGTGCAGGCAGCTGCGAGCCTGTGTATCCAGCAGAAGGATCAGGAAAGGATTCCTGGAGGAAGCGCTGTTCTAGCCAAGACCTACGGGGGCATTATTAACCAGGCAAAGGGGACGGTGTCCAAGCAGTGGAATGAACGTGGATTGAAGCTGTGAGGCAGGAGGGAGTGTGGCCTGTGCAGAAGGGACCGAGGCTGGTGAGACCAGGAGGGCCTGGGTGGCCTCCAGGTCAGATGTGAAAGGAAGAACTTGGCCACAGTCTGAGCTTCTCAGGCGTATGGCAGGGCTGCCTGGTGAGAGGGAATGAGCTCCCTGCTCTGGAGGTATGCAAGCAGGACTGGGCTCTCACCTGCCAGAGGCCACAGAGCTTTCCAGAGGCTGGAAGAGGCCACTCCAAGGCCTCTTTGCCCCTGAGAGTGGTGGCTCTTCTTGAGGCCACCTTGCCACGCTGTCACAGGGAACTAGCAGCCCCTGCCTCACCCGGGGGTTTGGAAGATAGAGGGAGGCCTAGGAAGGGCCCTGTGTCTCATCCGAGCTGGGCCCCTTTCCAGCCTCTCACTGGAAGGAAGCCCAAGGATGTTCCTGTGGGGGCTTTTACCAGGCCCACCTGCCCTCTGCTGGCCATGCTTGCAGCCTCCTGACCCTGTCCCAGCAGGACAGTGGGCTGGTGTGAGCGGGCAGGAACCGCCTGCACTTAGAAGGTGTGGGGCTGCCTCCCCGAGCTTCCATCTGCCGCTGGGGCCACACCCCAGGCCCAGGGATGGGACCCCACAGTGGTCACATCATCTTGCAGCAGAACCCAGGTACAGCTCCTGGAGCAGATGGTGGTCCCAAGCACGGGTGGGACCAGAAAGGACTCTCACCTGGGCTAACTCAGCTGCAGCCTCAGTTCCCTCCTCACACACGACGAGGAACATGGACTGGAAGCCTGCCCAGCAGGCCTTCTGCTCGATGTGCGTTGTGTGGCTTACGTCCAGGGAGGGAAGCAGCCTCTGTGCTGTCTTCTAGATAAGCCTGTATTCCCCGGGCTGTCTGCCAATGTATCCAGTTGTCCCGTCAGCCTGGAAGCTCTGAGGGAAAACCTTGGGCTGCTTCCTGAGCACCTGTATCCCCTGCAGCCAGCCCGGGGCCTCTGCTAGGAGCAGACTGAGCATGGCTTATGGGCCTGGCACCATCTGGCCTCTGCCCACCTTGCTGGCCTTGTCTTGTGTCTGCCCCTTCGACATTCCATAGCCCAGCTCAATATCTAGTGGTTCCTCTAGGGTGGCGAGCACTGTTTGGTCTCCAGATGTCTTCAGGTCGGAGCTCACAGCGCTCTCAGCCACCCCTTCCCAGTGTAGCACCGGGCACATGGTAGATGCCTATTGATGAGTGAAAGCTCCTAACACACTCAGAGAGCAAGGACTCCGCCTCATCCCACAGCCTGGGAGGAGAGGCAGACTGCCAAGGACCTGCTCAGCATGCTACAGAAGAAACCAAAGTGCCCACGGGACTGATCAGTGGAGCTTCCTGCCGAGACTGGAGGCCTTAGGGCAGGGTAGACAGTGTGTGTGCAGGCTGGGGACTCACAGTTCGGACTGTGCCCAGACCTACTAGCATAGTGGGTGGGTGGGAGGATGCGGGACTGGGGGCCGACCTTGCCTGAAATTCATGTGGGATCTCAGAGCAGCCACTGAATTGCTCTGTAGGGGGCTAAATAGTGGCCCCCACAGATACACACACCCAGACAGAGCCTGTGAGCCAGACCTTATTTGGAGAAAAGGTCTTTGTAGATGTAATTAAGCATCTCAAGATGGCATCATCTGGATTATGCGGTGGGCTGTAAGTCCTGTGATGTGTCTTTATGAGAGAAAGGCAGAGGGAGATTTGACACACACAGGAGGGGCCACGTGGAGACAGAGGTGGAGATTGGAGAAATGTGGCCACAAGCCAGGGAACACCAGCAGCCACCAGAAGCCGGAAGACGTGAGGCAGGGTTCTTCCCAGAGCCTTCGCTGCTGAGTCTGGGAATTTGTGACCGAAGCCATAAGAAGTGGGTACACGCCCTGAGCCTCCCACACTTGCTCACCTGTCCTGAGATGAGAATCTCTACTCTGCAGCATATTTGGAGGATCACTGCGGGGGCCACAGAGGTGCTGTTCAGATGGCACTTCAGAAGACTCAGGAGACCCTGGGGCAGGAGCAGTTTGACTGACAGCCCAGAGGGCTGCCCTCTGATTCCACCTGAGGCCCTGCTTTTCCTGGCTGCAGGGGTTCCAGGGCCAGGCCATTTCCGCTGGCGCAGGACTCTGCTAGCAGCAACCTGCCTGAAGTCTTCCTTTGGCCTGGCTGAGAGTTTCTGAGACCTGCGCTGGAGCGGAGGTGCTTCCTTCCTTGCTTCCTTTCTTCCTCTCTCCCTTCTCCATCCAGCAGGCTGGACCTGCCTGGCATCTGTGAGCTCTCCCTACTTTCTCCTATACCCTAACCTTTGTCCTGCATGGGCGACTCCCCCAGTGAGTCTCTTGCAGCTTTTACCCCAGTGCCTGCTTCTTGGAGAATCCAAACTGATCCAGTTAGGGATGATAAAGTGTAGGGTAGGCGCTCGGTGACTGTTTTCTCTGAGGTTGTGACTCGTGTGAGGCAGAAGCAGTCCCCGTGAGCCCTCCTGGTATCTTGTGGAGTGGAGAACGCTTGGACCTGGAGCCAGGAGGCCCAGACATACATCCTGTCCGAGCTGCAGCTTCCTGTCTCTAAAATGAGCCGGCCAGCGCAGGTGGCCAGACATCACTGTTATTCTCCTTTGAGTCTTTAAATCTTGTTGTCTTTCTTGCAGACTCGGTGAGCTGTGAAAGGCTATAATAGGGGCTTTATTTTACACTTTGATACTATTTTTTGAACATTCATATTATTGTTAGATATTGATATTCATATGAAGGAGCAGGATGACTTGGGTCCTTCTTGGCAGTAGCATTGCCAGCTGATGGCCTTGGACAGTTACCTGCCCTCTCTAGGCCTCCCTTTCCTTGTCTATGAAATACATTATAGAATAGGATGTAGTGTGTGAGGATTTTTTGGAGGTTAAACGAGTGAATATATTTAAGGCGCTTTCACCAGTGCCTGGGATGTGCTCTGTAGTTTCTGTGTGTTAACTATAAGGTTGACTTTATGCTCATTCCCTCCTCTCCCACAAATGTCGCCTTGGAAAGACGGAGGCAGCCTGGTGGAGGTGTATCTCCTAGACACCAGCATACAGAGTGACCACCGGGAAATCGAGGGCAGGGTCATGGTCACCGACTTCGAGAATGTGCCCGAGGAGGACGGGACCCGCTTCCACAGACAGGTAAGCACGGCCGTCTGATGGGAGGGCTGCCTCTGCCCATATCCCCATCCTGGAGGTGGGTGGGGACTGCCACCCCAGAGCGTTGCAGCTGTACTCCTGGGTTGCACCCCCCCCAGCTGTCACTGTCCCCTCCCTGCCATCAGTTGTGGGAAGGGCGTTCATCCATCCAGCCACCTGCTGATTTGTTATAGGGTGGAGGGGGGGTCTTTCTCATGTGGTCCTTGTGTTCGTCGAGCAGGCCAGCAAGTGTGACAGTCATGGCACCCACCTGGCAGGGGTGGTCAGCGGCCGGGATGCCGGCGTGGCCAAGGGTGCCAGCATGCGCAGCCTGCGCGTGCTCAACTGCCAAGGGAAGGGCACGGTTAGCGGCACCCTCATAGGTAAGTGATGGCCCCAGACGCTGGTCTCTCTCCATCTGGACCTGGCCTGGGAGGTGGCTTGGGCTGGGCCCAGGGAGAGCTAATGTCTCCTAACCAAGAATGCTGTGGCAGCCTCTGCCGCAGAGCCAGAGAACCAGAGTGCCAAGGCTGGCAGGGTTCCCAGTGGCCACGAGTGCAGATGAAGAAACCCAGGCCCCAAGAGGGTCATGCAGGTAGCCCAGGGAGTTCAGCCTTGACCCTGGGTCAATGACCTTTCCACAGTTCCACACTGCTCCCCTTTTAAAATCCGGTGATGTCTTTATGTCTTTTGTTATGTTATCTTCAATGTGGAGGGACTCGAGGTGATCTAAGCAAACTTTTTCTATCTTCTGCTTGCATACCTCTGAGACCAGGGGACTCACTCACTTGCATGACTGGGCCCTGCAGGTCACACTGGCCAGGCAGATGTGGTGGAGGAACTGGCAGAGGACTTTTTCTAGACTGTGACTACATTTAGTCCACCCAGCGGCCCCCCTATGAAGTCCAGTTGAGAACTAGGACTCTGGGGGCCGGTGGACAGAGAAGAGGGAGGGTTCTCTCCCTTACTGACTTCCTTCTGTGGCCAGACATTGAGCAAGGCCTCTGTACAGCATGTCCTGGGGCTGGCCTTGCCGTAGCTGCTAAATAGTTGACGAAACCAGTCCAGAGAGGGGAGGTGACTGCCAGGGTCGCACAGCTCAAGCTGGGGAACTCGCTGGGAAAACTGTCAGCTCTGGGCAGCAGCTTGACTTCCACTGTAAGCCCCAGCCCCCAGGGTCAAACACTGGCTCTGGTGCTGGCAGAGGCAGCCCACTAGCCTGTTTCAAAGGCTGAGAAGGCCCAGGAGTCTGCCCTGTGCTCCACCAGTTCTGCCCTGAGACTTTCCTACAGAGTACAGGTTTTGATGTTCAGTTTTAAAGGCAAGAATCAATAACCTTCTGCCCCATCAGGTGACCCCTTGTGCCTGTCCCACCCCTTTATTGACTGACCTCGGCTCAGTCAGGTCAGTTCCTGAAGGTCAGTGTGTGGAGGGGAGGCTGTTCTTTCCCAGAAAGGCCTTCCCCAGGCCTGGTGCTCTGGCCTCTGGAGGACTTCCTGGAGAAGTCCCTTCTTTGGGGTCCCAGTCAGTGTATGGGAAGCCCTTATTGCATGACCTGGCACGGGGCAGGGGCTCAACAGTCACTATTGCCTTCCTTGCCACTGCCATTTCCTCCTCTGTAAGCAGGTGATTGTGTGTCCAGTCTGAGCACAGAGATAAGCACACAGCAGGTGCTTAATAACTAGCAGCTGTAGGCTGGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGTTCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGTGGGTGTCTGTATCCCAGCTACTTGGGAGGCTAAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCAGTGAGCTGAGATCGTGCCACTGCAATCCAGCCTGAGTGATAGAGCGAGATTCCATCTCAAAAATAAATAAGTAAATAACTAGCAGCTGTAAATGTGGCTGTTGTTCTTCACCTCCACACTCAGTGCCACTCCACTCCCTCCCTCCGTGGTGTGAGGGGCCTCACTAGCTGTCTCCTAGGAGGAGCATGGCTGTGAGATTCCAGCTCCATCCTTGGCCACGGCTCCTGGAGACATCTTAGAGGCCAGGATCCAGAAGGCTCCCACACCTCATTTGACAGGGGAGAAGCTGTCAGTTCCAGGTCCCCTTGCACATCAGGGCCAGAGCTGCGTTAGGCCTCCAGTCTCCAGGCCACTGGGCCAGAGCTCACAGGCTGGCAGAGGGTTAGAACTGTTACTGGTGGCTGGGTGCAGTGGCTCACGCCTGTAATCTTAGCACTTTGGGAGGGCAAGGCGGGAGGATCATGAGGTCAGGACATCGAGACCATCCTTGCTAACACGGTGAAGCCCCGTCTCTACTAAAACTACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCACTCCAGCCTGGGCAATAGAGCGAGACTCCGTCTGGAAAGAAAAAAAAAAAAAAGAGCTGTTACTGTTGACAGTAGCATGAGGTAGACCATGGCCTGCACCAAAATGGGGGAGTGGAGTGCCACTGAGGCCAGAAGGAACCACACCCTCAAGGGTGGGGAGTTATGGTATGGGGGGTCCTAGGCATGGAGTCTTTTAATTCTTTAGACAATCCTGGGAGCAACTGTCCCTGTTTCACAGAGGGCGGGGCCACACAGCTGGTGAGTGGGCAGCCAAGACTCTGTTCAAGTTTGTGTGGGTCCAACACTTGCGGCCACGGTGGAGGGGCATCTGAGCCAGGCCTCAGAGAGTGGCGGGGGAAGTTGGGTGGGGAAGTGTGCCCTTCTCATTCCTCTGAGGCTCATCCTCTTGGTGCCTCTCTTTCATGGAAAGGGATAATAAGGTTATTGTGAGGATCCCCTGAGTTCGTATATTCAGACGCTTAGACAGAGCCAGGCACAGAGAAGGGCCCGGGGTTGGCTAGTTTGATTGCTGGTGTAATTGCTAATATCTTCCAGTTTGTATTGGTCAAGGTTCTGCAGAGAAGCAGAACCAGTAGGATGTATATATTAAGAGTTTCAAGCTCATGTGACCGTGCGGGCTGGCAAGTCTGAAATCCGCAGGGCAGGCCAGGCAGGCTGGCAATTCCTGCAGAATTTGATGTTGCAATACTGAGTCCTAAGGCAGTCCTGGGGCAGAATTCCTTCTTCCCTGGGAGGCCTCAGTCTGTTCTCTTAAGGCCTTCAACTGATTAAATGAGGCCTGCCCAAGTTATAGAGAGTAACCTGCCTTACTCCGTCTTCTGATTTAAATGTTAGTCACATCTAAAAAATATTTTCGCAGCAGCATTTCCACTGGCTTTTGACCAAACATCAGGCCACAAAGTTGATCCCCAAAATTAACCATCACTCTGTGCCTGTAAGGGAGGGGCTGGGAAAGGGGAGCAGGTCTCCCCAAGGGGTGACCTTGGCTTTGTTCCTCCCAGGCCTGGAGTTTATTCGGAAAAGCCAGCTGGTCCAGCCTGTGGGGCCACTGGTGGTGCTGCTGCCCCTGGCGGGTGGGTACAGCCGCGTCCTCAACGCCGCCTGCCAGCGCCTGGCGAGGGCTGGGGTCGTGCTGGTCACCGCTGCCGGCAACTTCCGGGACGATGCCTGCCTCTACTCCCCAGCCTCAGCTCCCGAGGTAGGTGCTGGGGCTGCTGCCCCAAGGCGCGGGTAGGGGGCGGAGGGCGGAGGGCGGAGGGAGGGCGGGCGGGCAGGCGGGCTTCTTGTGGCACGTGGGCTTCTTGTGGCACGTTCCTGGAGGCCGAACCCTTCTGGCTTTGGAAGGAGTCGTCAGAGACCCCCGCCATGCGGGAGGCTGGGGAGGAAGGGGCTCGAAACCTCCATCATCGCAGAGTCTGAATAGCAGTGGCCCCGCCATGCGCCCACGTAGCGGCGCCTACGTAGCCACGCCCCCACACCCCGTCCTGGCCACTCTCCCTCCTGAAGGTCTTCTGGTACCCGCCCCCTCCCCATCTCCATCCCCAGGCCCTGCGTCCTCTGCCCAATACTCTTTGGGCCTCCCTGTTGTCCAGCTCTCTCCGCGGCTCCATGACTGACAACTTGAGCAAGGCTAATGTGAATGGGAGCGGTTGAGGGCTCAGACCTCTCACCCGAGGAACATCCACAGAGTGTGCCGCATGCCCGGTGCAGTGTGGCTGCGGGGACACAGACACGGAGCCTCGGCCCTGAGGAGCTGGGGGGCAGTGACCGTCCCTCCTCTGACCCACCACTCCTCCAGTGTCAGGACACTGCGGGTATCTAGGGGAAGGAATCTTGTTCCACTTCAAGTCTGGAACTTCAAGTCTGTGTGTGTGCGTGCGCGCGCGCGCGTTGGGGGTGGGGGTTGCAGAGCAGATGCGTACCTGACAGCGGTAACCTAGGTCCCCCCTGGCCTATCAAGGCTTCCCTGGCGGCCGAATTTAAAGGCATCAAGCAAACAAAGCCCAACACATCTCTGCCTTGTCCTCTCAGTTTCCCCCCGTGGCACTTAGAACCACTTGATACACCGAATAGTTTCCTATCTCCCCCACTAGGATGTAAACTCCACAGGGGCATTGGGAATGCTGCCTGGCTATGGTAGGGACAGAGGGGAGCACCAGGGCGGGGCAGGGGTGCCAGAGTTCTGCCTGGGCAGTCAGATTTTCCTTAGGAGGGGACATTTGAGTGGGACCCAAACAGGTGTATAGCAGTTGTCCAGCCCAGCTGGCAAGGCCTGAGTCTGCCTCTGCAACCCCTCTCTTGGGCTCCTTTCTCTGCCACCCACCTCCTCACCTTTCCAGGTCATCACAGTTGGGGCCACCAATGCCCAAGACCAGCCGGTGACCCTGGGGACTTTGGGGACCAACTTTGGCCGCTGTGTGGACCTCTTTGCCCCAGGGGAGGACATCATTGGTGCCTCCAGCGACTGCAGCACCTGCTTTGTGTCACAGAGTGGGACATCACAGGCTGCTGCCCACGTGGCTGGTAAGTCACCACCCCACTGCCTCGGCCACCGTGATGCTAACAGCCCCTTTGGCAGTCAGGGTCTGTGCCGGGACCTCCAGTGCCAGGCTCTGTGCAGGGGGACCAGAGATGAAGTAGGCCTGATGGTGCCTTCAAGGACACTCAGTCTGATGAGGGAGGCGAGTGCACAGAGGAAACACGAGGTCAGGGCTGTATTAGAGGGAGCCCAGAGGAGGCACCTGCCCAGCCCGAGGGTCAGAGAAGGCATCTTGGAGGAGGGACATTTGATCGGGAGCTTGATGGATGAATAGGAGTTCACCTGGCCGATAAGACAGCAACTACCAAGGCTTAGAGGTGTGAGAGGAGGCTGTCTTACCTCACTGAGTAAGGACTGCAGGCGGCTTACCTTCGAGAAGAGAGCTTAGTGTCTGTGTGCACGTGTGTTTGTGTGTATGTGTGTGCGTGTGTGCACTGGCAGGAGTCCCCTGCTGGGGCAGGAGGGCCGGGCCATCACCATCTTTCACCATTCACCCCTGCACCAGGCATTGCAGCCATGATGCTGTCTGCCGAGCCGGAGCTCACCCTGGCCGAGTTGAGGCAGAGACTGATCCACTTCTCTGCCAAAGATGTCATCAATGAGGCCTGGTTCCCTGAGGACCAGCGGGTACTGACCCCCAACCTGGTGGCCGCCCTGCCCCCCAGCACCCATGGGGCAGGTAAGCAGGATGGCAGGGTGGGCAAGTCCAGGCTGGGGCTTGGGAGGTCTGTGTGACCTTGACAGTCTCTCCCTTCTCCCTTGTCTGTGTAAGGAGGATGACGCCACCTTAAATAGGATTAAATGAGAATGGGGCTCTGAAAGGGCTGTGCAATATTTTCATAACGTGTTTTTATAGAGACAGTTGAGTATGTTCTTTAAGCCCTCCTCTCTCCTACCATGAACTAAAGATTTCTGTGGAGGTCCCCTCACTCCCAGCACCCCCTCCTCATCCCAGGCCCTTTTTGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCCACAGCCGTCGCCCGCTGCGCCCCAGATGAGGAGCTGCTGAGCTGCTCCAGTTTCTCCAGGAGTGGGAAGCGGCGGGGCGAGCGCATGGAGGTGACTGTACCCCTCCTTCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCAGTGCTGGGCCCTCAGGGACCCCCAGCAAGCCCCTCCATCCTCCAGACTCCAGCTCTTCTGTAAGCTTACAGGGCTGGCCAGACCAGGAGTGGGGCACTCCTCACTTCACGCGGCTGGGGGCTGCTGGAGAGAGCCACAGCGGGAAGGGTTTCCTAGAGGCTGCAGGACAGTGCTGGATGGATTTTCAATGCTCACCTGGGTGTGAGCGTGCGGCAGGGCCGCGTGAGGGTCAGCGATCTGCTACTCTGGACTCAGCCATCTCTAGGCCCCTCTCACTCAGGTGCTCCATGGTTCTGGGAGCTGAGAAATCTCAAACCAGCAAAAAAGTGGAATTGATGTTGATGCTACAGGATAGTGCACAGATGCCATCTGGTTGCAGCATTTTGGTGGAAGGGCAGTGCCCAGCTAGGAGAGTGAGGAGGGGCAGGCATTTCTGGCTTGAGGAGATGGGGTCTTAATGCTCGTGTGAGAGGCAGAGTGGGTGGAGTGGAGCTGGCTGGATCCTTGCTTTGGCCTCCTGGATTTCTCTCTATCTCCATTTTGAAACCACTCTGTGTTTGGAAGAACTTTTGAGTATTCAGAGCTGCCCACTGGCAGAACAGTCTTCCTTGGGCAGGAGTGAGCTCCTTGTCCCCAGAAGGCTGGGTCTGGCTGGCCCCTGGCAGGGACACTGATGAGGGTGCTTGAGTTGATCCTGTCTAGTCCCTTTCTGTGTTTTCAAAGCCCATTCTAAAGCAGATTCCCATTTCCGTCTTTGACTCTAAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGTAGGAGGCTGGGCTTGCCCTGGGGTGAGGAGGGGTCTCTTTCTCCTTATGCACCCACTGCCCGCGAGGCTTGGTCCTCACAAGTGTGATCCATGAGACTCAAGCCTGACTTGCAGTTCCATACTCTGGTTCTGCCACTTCCATGCCCTTTGAGCCTGGGCAGGTGACCTTACTTCTCCTCATCTCAGCTTCCTCCTCCATAAGAGGGAAAAAGGTATTACCTGCCTCATTGTGTTGCAAGGAGATGGGCAGCATCTAGGGCACTGGCCTGGAGTATCGCAGGTGCTTTGCCTAAGGTGGTGCAGTCCAGGAGAGGCAGCTCCAGAGAGAGGCCCCCGGCTGGGGCTGAAAGGAGGGCAGACCTCGGTTTGAATTTCACCCTGCCGCTCTATAGCTGTGTGACTTGGGCAAATTACTTAACATCTCTGTATGAGGAAATGATGAGTGCTAAGCACTTAGCTTAGTGCCGGGACAATATAAATTCTAGCTATCGTTACTATTGTTTTCATCACCCGTTGCTTTAAAATCCAGCCTCTGGTATAGGCAACTATTGACGGGCTACCCTGTGTCGAAAACATGCCCAGGCAGGTAGCAGGAAGTCACAGATGGGGACCTCTTGGGGCATCAAGGGATGGTGCCCTGAGGCTGAGCTGTTCTGGTTGGGTGGAGCATGAGAGGTCTGGGAAGACAGTGGGACTCCAGCCTGGAATAAGAGGCTCAGAGTTGATTCTCGTCTGAGCACGTCCAGGGGAACCACTGAGGGTTTGGGAACAGGAGAGTGAGGGTGAGAACCTGGTTCTGGGCACAGCAGGCTGGCATGTAGGATGGATGTTCAGGAAAGATGAGCATAGTCAGGTGGCTGGTGCCCTTGTCCAGGGGAGAGGCTCCGTCAGGTTCAGGGGTCCTGGCTTGGAGGGAAGTCCGCCATGCTCTAATCACGCTCCCCTTTGGAAGTGCTCAGCCGATGAGCTCACAGGCACATGTCAGTTTGAAGTCATGGAATCTGACTCCATGAAGCGCACCTCAAAGAGCACCATTTTGCAGCTAAGGGAACTGCAGGCTGGACATGCTGAGTGGCTGCCCCGAGCCCTTGCAGCTAGGACATAGAGAATGCTAGTAACCACAACCCTACCATGTTCAGAGCACATGCCAGGCTCCATGCTGGGGCTTCGCACGTGTCATCTTCACAGTGTCCCTGTGAGTAGGTGTGGTTTCTCTTTCCATCTTACAAATGAGTAAACAGAGCCTCAGTGTAGCTAAGTAACCACTATTTTAGGTTTCTTAGCCAATGGGTGTGTCTGACTCCTAAGCCCATGGAGGGCATTCTGAGGTGGTTCAGACAGACCCCGGCTTACCCTTGAACTTCTGCCTGCTGGCTGCATAGGGAGGGGCTGGGGGGAGTTTGAGCATCTCAGGCCATAGAGCCCCTGCCTCACTGTCTCCATCTCTGGGTGGAAAGATGGTGTTTTCCCTGAGAAACTAAGGCTCAGAGAGGTTGAATGGCTCTCCCAAGGTCACACAGCTGGTCAGCTGCAGAGTTGAGAACACAGGAGTCCTGGTGCTCAGGCCAGCATCTCTTTTTTTCTTTGAGTTGTTTCTAGGTTTCCTAGCTCTTGCCTCAGACCTTAAAGAGAGAGGGTCTGATGGGGATGGGCACTGGAGACGGAGCATCCCAGCATTTCACATCTGAGCTGGCTTTCCTCTGCCCCAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAATCCCGGCCCCTCAGGAGCAGGTGAAGAGGCCCGTGAGGCCGGGTGGGTGGGGTGCTGCGTGTCTCTCCTGCACAGCTTTTCTGTGTCAGTTTGTGCCACCACCATACCGCCATGCATCAGGGTGGCGGTTTGCCAGGTAGATGCTGTGGGCAGCTTCCGCCATTGTGTGGACAGCATGTATATGTGTCTCTGTGTGGCTGGGTCTGTTTTTGCTTTTGTCCAGATCAGTAAGGTTTGCTACCTGGGTACCCCACTCCACTTGGAGTAGAATGTGCATAAATATGGCATAAAGAAATGCAATATGCATGCATTTATTGATTGATCTATTTTTTTCTGAGATGGGGTCTTGCTGTGTTGCCCAGGCTGGTCTCAAATTCCTGGGCTCAAGCAATCCTCTGGTCTCAGCCTCCCCAAGTGTTGGGATTATAGGCATGAGCCGCTGCACCTGGCCTCTCTGATCTATTTAACAAACCTGCTGGGAGGGTCTCAGGGTCAGGAGCAGCACTGGGCTCTGAGGACACAGAGCTCACTCAGCCGTGACCCAGAGGGGGTGCCTGAGCTGCATGCTGAAGGTTGTTAGCATGACCAGCAAGGCAAGAAAAGGCCCTGCCGAGATTAGCAAGGCATGTGCCAAGCCCTGGAATGTGACAGCCGGGCCTTCTAGAAACCTGAGTGTATAACTCTCCTTAAAAGCCAGTAGGAGCTCCTCAAAAGGCAGCCCTAAGGAGTCCACTCTTAAATGAACTCAGAGTCAGTTTTAAAATGCAAGTCTGTGTTGATTCTGGTCTGGATGGTGCATTCCTCGAGAGCAAAAGACAGTCTTGGTCTTGGATCCACTTGCCCTGGGTACACTGAGGGCTGCTAGGTTCCAGGTGCTCTTCCTGGCACTGGGGAGGGATACAGGCCCAAGAGACATGCTGTTCTCCCTCCTGGAGCATCTATTTTAGTGGAGGAAGACAGAAAACAAACCATTAATATAGAGTACTGAAAAGATGCGATGGAGAAAACTATAGCAAGGAAGGGAATGGGGTGGGAGAGAGGTCAGGAGAGGTCTCGCTGACAAGGTGGACGAAACAGGCCATGAGGCAGAGAACATGTTCCAGGCAAAGCAAAGGCCCCCAGGTGGGGATGTGCAGGGAGTACCAGGAAACCAGAGAGGTGGGAATAGTTATGAGATGGGGGGTGCCTCAGAGGGGACAGGGCCAAGTCAGGTGAGACCTGAGGGTCACAGTCAGCAGTGAGCTGGGGCCATGCAGGGGTCTGGCCTCAGAGGAGTGTGGTCTGGCCTGGATCTGAACCTCTCACTGTGGCCTAGCTGCTGAGCTGAGAAGAGATGACAAGGACCTTGGGCAGAAGCAGGGAGACTGGAGGGAGGCGGTGGAGGGTCCAGGCGTTGGGGCGGGGCTCAGGCTGGAGTCTGAAGGGAGCCTGCAGGCCTGGTGGGTGGATGTGGGTGGGAGAGGGGGAGGATGGCACCAAGGCTCGGGCCCCTGGACAGATGGAGTTGCCATTAAGTGGGATGGGGCAGGCTATGGGGCCATCAGTTTCAGAGGGATGAGTTTGGCACTGGCATGGTAGGCATCTGTCTATCTCCACGGCCCTCAAACCAGGCATGAAGCAGGAGCTCACGTGTTTGGTCAGCCATGGTGCAGAACCGCCTGGGTGGGAGGTGCGGGGTGGGAGATACACGGTTGTGTCCCAAATGGGCTCTGAGCCAGCGAGGGCCGTCTGCACTTTGGCCTCACAGAAGGATGTCGGAGGGAGAAATGAAGTGTGGGTGGGGGTCCCGGGCCACGCTAGACATGTGCTTTCTTTTCCTCGGGCTCTGGCAGGTGACCGTGGCCTGCGAGGAGGGCTGGACCCTGACTGGCTGCAGTGCCCTCCCTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCACTACAGGCAGCACCAGCGAAGGGGCCGTGACAGCCGTTGCCATCTGCTGCCGGAGCCGGCACCTGGCGCAGGCCTCCCAGGAGCTCCAGTGACAGCCCCATCCCAGGATGGGTGTCTGGGGAGGGTCAAGGGCTGGGGCTGAGCTTTAAAATGGTTCCGACTTGTCCCTCTCTCAGCCCTCCATGGCCTGGCACGAGGGGATGGGGATGCTTCCGCCTTTCCGGGGCTGCTGGCCTGGCCCTTGAGTGGGGCAGCCTCCTTGCCTGGAACTCACTCACTCTGGGTGCCTCCTCCCCAGGTGGAGGTGCCAGGAAGCTCCCTCCCTCACTGTGGGGCATTTCACCATTCAAACAGGTCGAGCTGTGCTCGGGTGCTGCCAGCTGCTCCCAATGTGCCGATGTCCGTGGGCAGAATGACTTTTATTGAGCTCTTGTTCCGTGCCAGGCATTCAATCCTCAGGTCTCCACCAAGGAGGCAGGATTCTTCCCATGGATAGGGGAGGGGGCGGTAGGGGCTGCAGGGACAAACATCGTTGGGGGGTGAGTGTGAAAGGTGCTGATGGCCCTCATCTCCAGCTAACTGTGGAGAAGCCCCTGGGGGCTCCCTGATTAATGGAGGCTTAGCTTTCTGGATGGCATCTAGCCAGAGGCTGGAGACAGGTGCGCCCCTGGTGGTCACAGGCTGTGCCTTGGTTTCCTGAGCCACCTTTACTCTGCTCTATGCCAGGCTGTGCTAGCAACACCCAAAGGTGGCCTGCGGGGAGCCATCACCTAGGACTGACTCGGCAGTGTGCAGTGGTGCATGCACTGTCTCAGCCAACCCGCTCCACTACCCGGCAGGGTACACATTCGCACCCCTACTTCACAGAGGAAGAAACCTGGAACCAGAGGGGGCGTGCCTGCCAAGCTCACACAGCAGGAACTGAGCCAGAAACGCAGATTGGGCTGGCTCTGAAGCCAAGCCTCTTCTTACTTCACCCGGCTGGGCTCCTCATTTTTACGGGTAACAGTGAGGCTGGGAAGGGGAACACAGACCAGGAAGCTCGGTGAGTGATGGCAGAACGATGCCTGCAGGCATGGAACTTTTTCCGTTATCACCCAGGCCTGATTCACTGGCCTGGCGGAGATGCTTCTAAGGCATGGTCGGGGGAGAGGGCCAACAACTGTCCCTCCTTGAGCACCAGCCCCACCCAAGCAAGCAGACATTTATCTTTTGGGTCTGTCCTCTCTGTTGCCTTTTTACAGCCAACTTTTCTAGACCTGTTTTGCTTTTGTAACTTGAAGATATTTATTCTGGGTTTTGTAGCATTTTTATTAATATGGTGACTTTTTAAAATAAAAACAAACAAACGTTGTCCTAACHuman PCSK9 Amino Acid Sequence (NP_777596.2) (SEQ ID NO: 6)MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ

Apolipoprotein C3 (APOC3)

In some embodiments, the target gene for modification using thecompositions and methods disclosed herein is gene encoding APOC3. TheLDL-R mediated cholesterol clearance pathway involves multiple players.Non-limiting examples of protein factors involved in this pathwayinclude: Apolipoprotein C3 (APOC3), LDL receptor (LDL-R), and IncreasedDegradation of LDL Receptor Protein (IDOL). These protein factors andtheir respective function are described in the art. Further,loss-of-function variants of these factors have been identified andcharacterized, and are determined to have cardio protective functions.See, e.g., Jorgensen et al., N Engl J Med 2014; 371:32-41 Jul. 3, 2014;Scholtzl et al, Hum. Mol. Genet. (1999) 8 (11): 2025-2030; DeCastro-Oros et al., BMC Medical Genomics, 20147: 17; and Gu et al., JLipid Res. 2013, 54(12):3345-57, each of which are incorporated hereinby reference. Thus, some aspects of the present disclosure provide thegeneration of loss-of-function variants of APOC3 (e.g., A43T and R19X),LDL-R, and IDOL (e.g., R266X) using the methods and compositionsdisclosed herein.

Apolipoprotein C-III (APOC3) is a protein that in humans is encoded bythe APOC3 gene. APOC3 is a component of very low density lipoproteins(VLDL). APOC3 inhibits lipoprotein lipase and hepatic lipase. It is alsothought to inhibit hepatic uptake of triglyceride-rich particles. Anincrease in APOC3 levels induces the development ofhypertriglyceridemia. Recent evidence suggests an intracellular role forAPOC3 in promoting the assembly and secretion of triglyceride-rich VLDLparticles from hepatic cells under lipid-rich conditions. However, twonaturally occurring point mutations in human apoC3 coding sequence, A23Tand K58E have been shown to abolish the intracellular assembly andsecretion of triglyceride-rich VLDL particles from hepatic cells.

Loss-of-function mutations that may be made in APOC3 gene using themethods and compositions described herein are also provided. Thestrategies to generate loss-of-function mutation are similar to thatused for PCSK9 or ANGPTL3 (e.g., premature stop codons, destabilizingmutations, altering splicing, etc.).

In some embodiments, the gene modification methods and compositionsdescribed herein reduces expression of functional APOC3 protein encodedby the APOC3 gene in the cell. In some embodiments, the modificationreduces expression of functional APOC3 protein encoded by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, at least 99.5%, at least 99.9%, or 100%. In some embodiments,the modification reduces expression of functional ANGPTL3 proteinencoded by the APOC3 or ANGPTL3 gene in the cell by at least 2 fold, atleast 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, atleast 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, atleast 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, atleast 90 fold, at least 100 fold, at least 200 fold, at least 300 fold,at least 400 fold, at least 500 fold, at least 600 fold, at least 700fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least2000 fold, at least 3000 fold, at least 4000 fold, at least 5000 fold,at least 6000 fold, at least 7000 fold, at least 8000 fold, at least9000 fold, or at least 10000 fold. In some embodiments, the modificationabolishes expression of functional APOC3 protein encoded by the APOC3gene in the cell.

The protein sequence of human APOC3 can be found, for example, atDeposit No. NP_000031.1, which reference is incorporated herein in itsentirety. Human nucleic acid sequences can be found at e.g., GenBankAccession Nos.: NG 008949.1, which sequence is incorporated herein inits entirety. Mouse, rat and monkey APOC3 nucleic acid sequences havebeen deposited; see, e.g., Ensembl accession number ENSMUSG00000032081,ENSRNOG00000047503, and ENSMFAG00000001837 respectively, each of whichsequences is incorporated herein in its entirety.

The polypeptide and coding nucleic acid sequences of APOC3 and of othermembers of the family of human origin and those of a number of animalsare publicly available, e.g., from the NCBI website or ENSEMBL website.Examples include, but are not limited to the following sequences, eachof which sequences are incorporated herein in their entireties;

NG_008949.1:5000-8165 Homo sapiens apolipoprotein C3 (APOC3),RefSeqGene on chromosome 11 (SEQ ID NO: 62)CTGCTCAGTTCATCCCTAGAGGCAGCTGCTCCAGGTAATGCCCTCTGGGGAGGGGAAAGAGGAGGGGAGGAGGATGAAGAGGGGCAAGAGGAGCTCCCTGCCCAGCCCAGCCAGCAAGCCTGGAGAAGCACTTGCTAGAGCTAAGGAAGCCTCGGAGCTGGACGGGTGCCCCCCACCCCTCATCATAACCTGAAGAACATGGAGGCCCGGGAGGGGTGTCACTTGCCCAAAGCTACACAGGGGGTGGGGCTGGAAGTGGCTCCAAGTGCAGGTTCCCCCCTCATTCTTCAGGCTTAGGGCTGGAGGAAGCCTTAGACAGCCCAGTCCTACCCCAGACAGGGAAACTGAGGCCTGGAGAGGGCCAGAAATCACCCAAAGACACACAGCATGTTGGCTGGACTGGACGGAGATCAGTCCAGACCGCAGGTGCCTTGATGTTCAGTCTGGTGGGTTTTCTGCTCCATCCCACCCACCTCCCTTTGGGCCTCGATCCCTCGCCCCTCACCAGTCCCCCTTCTGAGAGCCCGTATTAGCAGGGAGCCGGCCCCTACTCCTTCTGGCAGACCCAGCTAAGGTTCTACCTTAGGGGCCACGCCACCTCCCCAGGGAGGGGTCCAGAGGCATGGGGACCTGGGGTGCCCCTCACAGGACACTTCCTTGCAGGAACAGAGGTGCCATGCAGCCCCGGGTACTCCTTGTTGTTGCCCTCCTGGCGCTCCTGGCCTCTGCCCGTAAGCACTTGGTGGGACTGGGCTGGGGGCAGGGTGGAGGCAACTTGGGGATCCCAGTCCCAATGGGTGGTCAAGCAGGAGCCCAGGGCTCGTCCAGAGGCCGATCCACCCCACTCAGCCCTGCTCTTTCCTCAGGAGCTTCAGAGGCCGAGGATGCCTCCCTTCTCAGCTTCATGCAGGGTTACATGAAGCACGCCACCAAGACCGCCAAGGATGCACTGAGCAGCGTGCAGGAGTCCCAGGTGGCCCAGCAGGCCAGGTACACCCGCTGGCCTCCCTCCCCATCCCCCCTGCCAGCTGCCTCCATTCCCACCCGCCCCTGCCCTGGTGAGATCCCAACAATGGAATGGAGGTGCTCCAGCCTCCCCTGGGCCTGTGCCTCTTCAGCCTCCTCTTTCCTCACAGGGCCTTTGTCAGGCTGCTGCGGGAGAGATGACAGAGTTGAGACTGCATTCCTCCCAGGTCCCTCCTTTCTCCCCGGAGCAGTCCTAGGGCGTGCCGTTTTAGCCCTCATTTCCATTTTCCTTTCCTTTCCCTTTCTTTCTCTTTCTATTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCCTTTCTTTCTTTCCTTTCTTTCTTTCCTTTCTTTCTTTCTTTCCTTTCTTTCTCTTTCTTTCTTTCTTTCCTTTTTCTTTCTTTCCCTCTCTTCCTTTCTCTCTTTCTTTCTTCTTCTTTTTTTTTTAATGGAGTCTCCCTCTGTCACCTAGGCTGGAGTGCAGTGGTGCCATCTCGGCTCACTGCAACCTCCGTCTCCCGGGTTCAACCCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCACGCGCCACCACACCCAGCTAATTTTTGTATTTTTAGCAGAGATGGGGTTTCACCATGTTGGCCAGGTTGGTCTTGAATTCCTGACCTCAGGGGATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGCGCCTGGCCCCATTTTCCTTTTCTGAAGGTCTGGCTAGAGCAGTGGTCCTCAGCCTTTTTGGCACCAGGGACCAGTTTTGTGGTGGACAATTTTTCCATGGGCCAGCGGGGATGGTTTTGGGATGAAGCTGTTCCACCTCAGATCATCAGGCATTAGATTCTCATAAGGAGCCCTCCACCTAGATCCCTGGCATGTGCAGTTCACAATAGGGTTCACACTCCTATGAGAATGTAAGGCCACTTGATCTGACAGGAGGCGGAGCTCAGGCGGTATTGCTCACTCACCCACCACTCACTTCGTGCTGTGCAGCCCGGCTCCTAACAGTCCATGGACCAGTACCTATCTATGACTTGGGGGTTGGGGACCCCTGGGCTAGGGGTTTGCCTTGGGAGGCCCCACCTGACCCAATTCAAGCCCGTGAGTGCTTCTGCTTTGTTCTAAGACCTGGGGCCAGTGTGAGCAGAAGTGTGTCCTTCCTCTCCCATCCTGCCCCTGCCCATCAGTACTCTCCTCTCCCCTACTCCCTTCTCCACCTCACCCTGACTGGCATTAGCTGGCATAGCAGAGGTGTTCATAAACATTCTTAGTCCCCAGAACCGGCTTTGGGGTAGGTGTTATTTTCTCACTTTGCAGATGAGAAAATTGAGGCTCAGAGCGATTAGGTGACCTGCCCCAGATCACACAACTAATCAATCCTCCAATGACTTTCCAAATGAGAGGCTGCCTCCCTCTGTCCTACCCTGCTCAGAGCCACCAGGTTGTGCAACTCCAGGCGGTGCTGTTTGCACAGAAAACAATGACAGCCTTGACCTTTCACATCTCCCCACCCTGTCACTTTGTGCCTCAGGCCCAGGGGCATAAACATCTGAGGTGACCTGGAGATGGCAGGGTTTGACTTGTGCTGGGGTTCCTGCAAGGATATCTCTTCTCCCAGGGTGGCAGCTGTGGGGGATTCCTGCCTGAGGTCTCAGGGCTGTCGTCCAGTGAAGTTGAGAGGGTGGTGTGGTCCTGACTGGTGTCGTCCAGTGGGGACATGGGTGTGGGTCCCATGGTTGCCTACAGAGGAGTTCTCATGCCCTGCTCTGTTGCTTCCCCTGACTGATTTAGGGGCTGGGTGACCGATGGCTTCAGTTCCCTGAAAGACTACTGGAGCACCGTTAAGGACAAGTTCTCTGAGTTCTGGGATTTGGACCCTGAGGTCAGACCAACTTCAGCCGTGGCTGCCTGAGACCTCAATACCCCAAGTCCACCTGCCTATCCATCCTGCGAGCTCCTTGGGTCCTGCAATCTCCAGGGCTGCCCCTGTAGGTTGCTTAAAAGGGACAGTATTCTCAGTGCTCTCCTACCCCACCTCATGCCTGGCCCCCCTCCAGGCATGCTGGCCTCCCAATAAAGCTGGACAAGAAGCTGCTATGA NP_000031.1 Human apolipoprotein C-III precursor (SEQ ID NO: 63)MQPRVLLVVALLALLASARASEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQARGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVRPTSAVAA 

Angiopoietin-Like 3 (ANGPTL3)

In some embodiments, the target gene for modification using thecompositions and methods disclosed herein is gene encoding ANGPTL3.ANGPTL3 has been associated with diseases and disorders such as, but notlimited to, Arteriosclerosis, Atherosclerosis, Cardiovascular Diseases,Coronary heart disease, Diabetes, Diabetes Mellitus,Non-Insulin-Dependent Diabetes Mellitus, Fatty Liver, Hyperinsulinism,Hyperlipidemia, Hypertriglyceridemia, Hypobetalipoproteinemias,Inflammation, Insulin Resistance, Metabolic Diseases, Obesity, Malignantneoplasm of mouth, Lipid Metabolism Disorders, Lip and Oral CavityCarcinoma, Dyslipidemias, Metabolic Syndrome X, Hypotriglyceridemia,Opitz trigonocephaly syndrome, Ischemic stroke, Hypertriglyceridemiaresult, Hypobetalipoproteinemia Familial 2, Familialhypobetalipoproteinemia, and Ischemic Cerebrovascular Accident. Editingthe ANGPTL3 gene using any of the methods described herein may be usedto treat, prevent and/or mitigate the symptoms of the diseases anddisorders described herein.

The ANGPTL3 gene encodes the Angiopoietin-Like 3 protein, which is adeterminant factor of high density lipoprotein (HDL) level in human. Itpositively correlates with plasma triglyceride and HDL cholesterol. Theactivity of ANGPTL3 is expressed predominantly in the liver. ANGPTL3 isassociated with Dyslipidemias. Dyslipidemias is a genetic diseasecharacterized by elevated level of lipids in the blood that contributesto the development of clogged arteries (atherosclerosis). These lipidsinclude plasma cholesterol, triglycerides, or high-density lipoprotein.Dyslipidemia increases the risk of heart attacks, stroke, or othercirculatory concerns. Current management includes lifestyle changes suchas exercise and dietary modifications as well as use of lipid-loweringdrugs such as statins. Non-statin lipid-lowering drugs include bile acidsequestrants, cholesterol absorption inhibitors, drugs for homozygousfamilial hypercholesteremia, fibrates, nicotinic acid, omega-3 fattyacids and/or combination products. Treatment options usually depend onthe specific lipid abnormality, although different lipid abnormalitiesoften coexist. Treatment of children is more challenging as dietarychanges may be difficult to implement and lipid-lowering therapies havenot been proven effective.

ANGPTL3 is also known to cause hypobetalipoproteinemia.Hypobetalipoproteinemia is an inherited disease (autosomal recessive)that affects between 1 in 1000 and 1 in 3000 people worldwide. Commonsymptoms of hypobetalipoproteinemia include plasma levels of LDLcholesterol or apolipoprotein B below the 5th percentile which impairsthe body's ability to absorb and transport fats and can lead to retinaldegeneration, neuropathy, coagulopathy, or abnormal buildup of fats inthe liver called hepatic steatosis. In severely affected patients,hepatic steatosis may progress to chronic liver disease (cirrhosis).Current treatment of hypobetalipoproteinemia includes severe restrictionof long-chain fatty acids to 15 grams per day to improve fat absorption.In infants with hypobetalipoproteinemia, brief supplementation withmedium-chain triglycerides may be effective but amount must be closelymonitored to avoid liver toxicity. Another option for treatinghypobetalipoproteinemia is administration high doses of vitamin E toprevent neurologic complications. Alternatively, vitamin A(10,000-25,000 IU/d) supplementation may be effective if an elevatedprothrombin time suggests vitamin K depletion.

In one example, the target tissue for the compositions and methodsdescribed herein is liver tissue. In one example, the gene is ANGPTL3which may also be referred to as Angiopoietin 5, ANGPT5, ANG-5,Angiopoietin-Like Protein 3, Angiopoietin-5, FHBL2, and ANL3. ANGPTL3has a cytogenetic location of 1p31.3 and the genomic coordinate are onChromosome 1 on the forward strand at position 62,597,487-62,606,159.

Loss-of-function mutations that may be made in ANGPTL3 gene using themethods and compositions described herein are also provided. Thestrategies to generate loss-of-function mutation are similar to thatused for PCSK9 (e.g., include, but are not limited to premature stopcodons, destabilizing mutations, altering splicing, etc.).

In some embodiments, the modification reduces expression of functionalANGPTL3 protein encoded by the ANGPTL3 gene in the cell. In someembodiments, the modification reduces expression of functional ANGPTL3protein encoded by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least99.9%, or 100%. In some embodiments, the modification reduces expressionof functional ANGPTL3 protein encoded by the ANGPTL3 gene in the cell byat least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, atleast 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, atleast 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, atleast 80 fold, at least 90 fold, at least 100 fold, at least 200 fold,at least 300 fold, at least 400 fold, at least 500 fold, at least 600fold, at least 700 fold, at least 800 fold, at least 900 fold, at least1000 fold, at least 2000 fold, at least 3000 fold, at least 4000 fold,at least 5000 fold, at least 6000 fold, at least 7000 fold, at least8000 fold, at least 9000 fold, or at least 10000 fold. In someembodiments, the modification abolishes expression of functional ANGPTL3protein encoded by the ANGPTL3 gene in the cell.

In some embodiments, a splice site disruption generated by a base editorsystem disclosed herein can result in the inclusion of intronicsequences in messenger RNA (mRNA) encoded by the ANGPTL3 gene. In someembodiments, the splice site disruption generates a nonsense,frameshift, or an in-frame indel mutation that result in premature stopcodons or in insertion/deletion of amino acids that disrupt proteinactivity. In some embodiments, the splice site disruption generatesexclusion of exonic sequences. In some embodiments, the splice sitedisruption generates exclusion of exonic sequences that results innonsense, frameshift, or in-frame indel mutations in the ANGPTL3transcript. Canonical splice donors comprise the DNA sequence GT on thesense strand, whereas canonical splice acceptors comprise the DNAsequence AG. Alteration of the sequence disrupts normal splicing. Splicedonors can be disrupted by adenine base editing of the complementarybase in the second position in the antisense strand (GT to GC), andsplice acceptors can be disrupted by adenine base editing of the firstposition in the sense strand (AG to GG).

In some embodiments, a base editor system provided herein effects an A•Tto G•C alteration in a ANGPTL3 gene when contacted with the ANGPTL3gene. In some embodiments, the A•T to G•C alteration is at a splicedonor site of the ANGPTL3 gene. In some embodiments, the A•T to G•Calteration is at a splice acceptor site of the ANTPTL3 gene. In someembodiments, the A•T to G•C alteration results in an aberrant ANGPTL3transcript encoded by the ANGPTL3 gene. In some embodiments, the A•T toG•C alteration results in a non-functional ANGPTL3 polypeptide encodedby the ANGPTL3 gene. In some embodiments, the A•T to G•C alteration isat a 5′ end of a splice donor site of an intron 6 of the ANGPLT3 gene.

The nucleotide sequence of human ANGPTL3 is provided, for example, inNG_028169.1, which is incorporated herein in its entirety. The proteinsequence of human ANGPTL3 is provided, for example, AAD34156.1, which isincorporated herein in its entirety.

Mouse, rat, and monkey ANGPTL3 nucleic acid sequences have beendeposited; see, e.g., Ensembl accession number ENSMUSG00000028553,ENSRNOG00000008638, and ENSMFAG00000007083 respectively., each of whichsequences are incorporated herein its entirety.

The polypeptide and coding nucleic acid sequences of ANGPTL3 and ofother members of the family of human origin and those of a number ofanimals are publicly available, e.g., from the NCBI website or ENSEMBLwebsite. Examples include, but are not limited to the followingsequences, each of which sequences are incorporated herein in theirentireties;

NG_028169.1 Human angiopoietin like 3 (ANGPTL3), RefSeqGene on chromosome 1 (SEQ ID NO: 7)AATGACAAACTGAAAAAATCTATTGTTTGTTATATATATAACAAAGAATTAGTATCCACAATATGTAAATAATTCCTAAAATTAGTCAGAAAGAGACAAACTTAAAAAGAGGGTAACAAGGAGGGGAGCAAATTATGTACATAACCAGATGATTCGCAAAGACGGCAACAGAGATGGCCAGCAAAACAAACTAGATATATACTTGTCTATTAGATTTATCAACATTTTTTGCCTTTTTCATTAAAAGCATTTGTAAAAGGATATAGGAAAAGAGGAACTCTCATATACTCCTGGCAGGGATGTAAATTGGTACAACCCTTTTGAAGGACAATCTGACAAAAGCAATCGTAAGTTACAAGTCAACATCTATGAATGTATATGAAAATATTTATATACATACATCACCACCATAAAAGCATTTTCTATACATACTGTTTATAATTAGAAAATTGGAAACAAATGATTAAAAGGGGGCTGATTAAATTAAGGTTCATCTATATAACAGGATTATGCAGCTATTAAAAAGGACGTGGTAACTCTATAGACATTCATAGGAAAATAAATTTTAAAATACTAAGATCCTGAATGATATATATATCATGAGCTATTATACATAACAAGATCCCACTTGTGTTATAAAAAATTATGTTTAGTCATTCAAAGGGTCTGGTATGATAGACCCAAAATGTTAATAGAGTCGAGATTTTTATTTTTTATAGGTTTTTGAAATACCTGAATTTTCACAATAAGTACTTTGCACATTAAAAATCTTAGCTGGGCATGGTGGCTCACGCTTGTAATCCCAGCACTCTGGGAGGCCAAGGTAGGCAGATCACCTGAGGTCAGGAGTTCGAGACCAGTCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGCTTGGTTGGGGGTGCCTGTAATTCCAGATACTCGGGAAGCTGAGACAGGAGAATCGCTTGAACCCAGGAGGGGGAAGTTGCAGTGAGCTGAGATCACGACGCTGCACTCCAGCCTGGGCAACAAAGAGCAAAACTCCGTCTCAAAAATAAATAAAGAAAAAATCTTTACATGTCCAAAGATACGGCTGTTCAACTAAAAAATATATATGTATAAAACTTAACATGTTAATAGTGAACACACAAAACAGTAAGATAGATAAAATTATTCCTTCAAAGCTCACTTAACCTCTGGATCTACACTGTCCAAAAAGACGGTCTAATGAGACAATTGAGCACTTGATAGGTGAGTGGTTCTAACTGAGATATGTTCTCAGTATAAAACATACAATAGGATCTTCCTATACAACATTAATTAAAAAACAAACTATTGTAGTTAAAAAGGAAAAAATTAGAGATACTATGTAAAAAAGAGCCAAAATACCTTGTATTTTATTTGAAAGACATATCTCCATAAGATTACACAACCTCGTGTAGGATAAAGGACTTTGCTTTGCTTGGAATTTAAACAATTTAGGCTCTTAAATGTCCTAAAATTCTCTGTAGCTAAGAAATTTTTATATTGGTTCCTAGGAACTAGGAATCCTTAAATTAGGCCCTACATTTGCTTACAAGTTTATTTTCCTTGGCATAAAATTTTTTAGTTTTTACATTACTGGTTATATTTGATCAGGGTTCTATTTAAATAGGCACAAGTTCAAGCAAAGATCAGATTCTGCTTTTAGCAGTGTGTACTCAGACAGGAAGTATTAAAAGGCAGGCAGAAAATCCTTTATAAAATTACTACTTTCAATGCATTTTCCCACGTTGAAATGCTTCTGCAGTTTATAATTAGGCAAATTACTTTAATTATAATCAATAATGCTGTTCAAATTACTATAAGAATTATACAGATATTTATACCAAGAGACAATATACTAGAAACCAAGACTACGTGACCATTACCTCTACTCTGTCAGTGTTATTTGTGAGAAATTGCACAAATTTTGCAAAAAGTGTTAGTATCCTACTACAGTAGGATATAATATAGAAGGAAATAATTTCATAAAGCCTGTCTTTGGTACTAGTGCTCAGTTACTTTCATTAACTAAAAAAGGGGCTACTCTTCAAATTCCCTTCTCTAAAAAGAATGTACTATATCAAAAGGGGGTAAACACTACTACGTATACATTCTGCACTTAGAAATCCCTATATGTTGATTTTATCATTCTCTTATTCAATAAATATTGTTTCTACAATGTGTAAGGCATTACTGTACTAAAGCATTATAAGGAATATAAGTTAAAAACACATACAAATCTTGCCAATCAACAGCTTATAGTGTAATAGGGGAGAGAAGCTGGCCCATCTATATTCTCCCTCAACTAGCAAGTGGATGAAATATCAGGGTCAATAGTTATAAGCCACAAAAGCTGACAGCTTAATTAAGAGAAGTTTTGAAATATGTATTTCATGACCAATAATTACAACTGTAACTTTTCTATTTAAAGAAGGAGAAAATTTGAATTTCTTCTCTAGCTCAACATACACTTCTATAATTCCATTACATGAACCAGAGTAAAGGGTAAGATGGAAATGAAGAATATTTTCTTACCCTTTTGTGGTTCTATATTGGACACTTAAAAATCATACACAACCTAATCAAAAGATGTAATTCTTTAAAAAGGTACGAGACCAAAATTCAGAAAATCTAGACTATAACAAAATTTCTCAATTTACATTATCTTAATATGCAATTAATTTTCACCAGTAAAATACTATAGTATGGGTACAAATGCATTGATTAGTTCTAATTACAAAAATGGCTAATATATAATACTGTGTAGTGTTTATGATACATCAGATAATGTTCTAAGTGCTCTGAAAATATAAACTTTTAATCTTTTATACGACCCTATAAAATAGGTGTTATTCTCACTGGAGAGATGAGAAAACAGGGGTTCAGAGATGTGAAGTAATTTGACCAAAGGTCACAAAGCTGAAGAATATGAAATCCGGGATTCTGATTCAGGCAGTCTTATTCCAGAATCATGCTCTTAACCACTATGGAATACTGCCTCTACTGTAACTATTATACCCAAAACCCTTAATCCTAAGTCATCAAAAGGAAGAGCCTCTATTTTACACAATGAAGAGGCATTTCTAAGAATAGAAATTTAGGGACGAGCACAGTGGCTTACTCCTTTAATATCAGCACTTTGAGAGGCTGATATGGGAGGTTCACTTGAAGTCAGGAGTTCAAGGTCAGCTTGGGCAACATAGTGAAACACAGTCTCTACAAAATATTTAAAAATTAGCTGGGTGTGGTGGCATGCATCTATAGTCTCAGCTACTTGGGAGACAGAGGGAGGAGGCTTGCTCGAGCCCAGGAGTTCGTGGCTATAGTGAGCTATGATCATGCCACTGCACTCCAGCCTGGACAACAGAGCAAGACCCTGTCTCTAAAAAAGAAAAGAAATTTGGAAATGGTTTATTTTGTATTAACAATTTATAATTTACACTGAAATTTATTATGATAAAACTTTTCCCTGTGTTAAAAAGCTATTAACTTTATGAAAAATTTCTTTTAGGTAAGGTTGATTATATATACCCACACACATACACAGGTTAAAAGTTAGTTTCATGTGACATAATAACTAGCATTTTGAGCACTACCTGTTTGCCCAGCACTGTTCTAAGTGCTCTACATGTATTATTGTTAAATTATCATAACACTATGAATTATGTACTATAATTACCCCAGCTTTACAGATGAGGAGACTAATCCATGGGGAGGTTAAGTAACTTGTCCAAGGCCAGACAGCTAGAGCCGGCTTTTGGACCCACACCACAGTCTGACTCCAGCACCCATATTCTTAACAATTTCACCATATTAATATGTCAAGATTAAGCAGTTTTAAAGGATGCTATTTTCTCACAAATTTCTTAATATGAACACTCAATAAGAATAATCACTAATATAAGCATTTAGTATTTTTTTAACACTAAGTTGGAAGCATAGTGGAACATTTATTTTTAGAAATATTATTAATTGGCTGGGCTCACGCTTGTAATCGGCTGGGCTCATGCCTGTAAATTTTGGGAGGCCAAGGTAAAAGAATTGCTTGAGCCCAGTATTTCCAGACCAGCATGGGCAATACATTAAGACATCATCTTTAAAAAAAAAATGTTATTAATCTCCTCTTTTTGTTAAATGTATATTATCAAAATTGTTACTAAGCTAACAAACTTCAGAAAAACTTATGATGGGCAAGCTGCTTGTGACATTGAAGGTATTTAAGATTCAATTCTAGTTTGGTCCTAGATGACCACATATCCATTGTTCCTTCAACGAGCACATGGTAAAGAGCCTAGAACACAGAGACACAGAACACAGTGGAGAAAAGGGAGTGAAATGTCTTTAATGACACTTACTATATATGGGATTTTGTGACAATATACAAGGATGGTTAAGACATATAAGGTGATGCAAAAAAACATATTAACAATTATAGTGACAAAAAATGAGGAGCATATAATTATACATTGATTTATACAGAGTACCAGAGGAACACAGCATTGAGAGCCGTAACACCACCTGAGGGAGTGGAGAAAGGCTTCAGAGAGAAAGTGTTTTTTGGAATGGATCACTGTTTCCAAAAGAACTAAAGTACAGTTTGAGAAATGCATACTTAATTCATTACTTTTTTCCCCTCAACTTTAATAATAAATTTACCCAACAAAAAAGTTTATTTTTGACTTGTAAATCTCTTAAAATCATAAAAAAGTAAAATTAGCTTTTAAAAACAGGTAGTCACCATAGCATTGAATGTGTAGTTTATAATACAGCAAAGTTAAATACAATTTCAAATTACCTATTAAGTTAGTTGCTCATTTCTTTGATTTCATTTAGCATTGATCTAACTCAATGTGGAAGAAGGTTACATTCGTGCAAGTTAACACGGCTTAATGATTAACTATGTTCACCTACCAACCTTACCTTTTCTGGGCAAATATTGGTATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATTGAAAATCAAGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACACCCAGAAGTAACTTCACTTAAAGTAAGTAGAAAATAAAGAGGGTTCATGTTTATGTTTTCAATGTGGATCTTTTAAAAAAAATATTTCTAAGGCATGCCATTTGAAATACTTTGTTGCATTGTTGAAATACTTTTTTTTCCAAGAAAAATAATCTCCAGAAAATAAAATTTCCTATTATAATTTCAAGTTAGTTTTTTGTTTCCCTAATGTTATATATGAAAACACTGAAAATTTGCATTTTATATGAAAATTACAAATCGGTTAAATTATACAATCTAGAACACTATGTCATTACACTATTGTAAATTACTGAAGGTAAGTAAAAAGTTAAAAAAAATTTAAAACTATTCTCCAGTGTTTAAAACAGATTAAATAATACAGTAAATGGAAAAGATTTATTCATATGAAAATATGCTGGGCTTTTTCTTTTAATTGAAGTTCAGAAAATCAAATTTTAGAGATAGTACAATTTAAATAAAATGTTAAGGACAAAAATATGTGCTATTTGAAAGAAGCATACAAGGGGAAGGAATTGCCAATATTCATTTTTCAAATCCATTATTAGTTTAAAAATTTAGATTATGATAGTGTTACAGGAAATTAATAGAAAAGAAAGAGGAAAGCAACTTATAACCAACCTACTCTCTATATCCAGACTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAACCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGGTAAGTCAGTATTTTAATGGTATGTCCCATCTTTCACACAGGTCTGTAAAAACACTGAATCCTAAAATTATTTACAAGCTTTAACTGGATCATGAGTAAAATTATCACATCAGCATAACTGTTAAAATTGCAGGCTCTGAAGCTAATAAACTACCTGCATTTAAACCATGGCTCTAAAACTTTGTGTGACCTTGAATAAATTACTTCACCCCTTTATCTCTCAGTTTCCTCACATATACTACAAAGATAATAACAGAACTTATAGGATTATTGTAAGAAAAAAAATTAATTCATAGCAGCCAATGTCATCTTACTAAAATTCAAATTAGATCATGTTTCTCTTTGCTCAAAACCACACAATAGCTTTCCATTTCACTCATATTGGCTCTTTAGACCAAGATTACCCAACCCTTCGTCATCTCACTGACTTCACCTCCTCTACTCTAGTTATTCTGACCGCTTTACCAGTATTCAAACACATCAAACATACTGCCACCTCAAAGCCTTTGCCCTTGTTGTTTCCTCTAACTGGAACGCTCTTCTGCCCTGGTATCTACGTGGCCCACTCTCTGATTTCCCTTAGGGTCGTTATCAAACAAAAAATTCCCAATGAAGACTTACAAGGTCACTTAACCAAAAATCACAACCGCCTGGTCCCATCCCTGAAAACTTCTACTTCCTTAGCTACTTTTCTCCTGCACACTCACCTTTATTTAACATAACATAAATTTTAGTTATTTATCTCTTCTATTCCTGCACTAAAATGTAAGCTCTGTGAATACAGGGATTTTTTCCATTATCTTCATATTTTCCATTATTTGTATATACTCCAGAATATAGAATACTGTATGGCACACAGTAGGCATTTCTGTTGAATTAATAAATGTAATGTCATATTCACACAGAAGCGTGTGCTATGATTATTATTACTTGGATTACTAGAAATAGTGTGCCTCATAATTAAAGGTCAACATTCAACAATGTAATTAATCTACAATGTAAACATCTGGTGAAGTGACAGAGGGAAGCACTTGTTTAGAAAAAAGCTATGTCAGAATCCATGTATTCTAATATGCAGTACAATAGTTTAAAAATATTAATAATACTCTCAAACAGCTATTCAAGAGGATTCAAAAAACATAATATAAACTCAGAGAAACTGGTAAACAAAATCATTTTCAAGAGATATAAAACAAATATTATTACCAATTTCCACTAAACAAACATAATGTTAGTAGTGCTGCTAAAAGGTTTTTTATCAACTACTTTTGGTTTCCATACTTTCCTTCTTATGATGTTATTATTCTAAATTCTTTTCAATTATATCTTTTACTATGATTAAATGAACCTGCTCCCCAAAGCAAAATGTTACTATAGTAATATACATTGTGTCTAAAAATAAAAATGTGTGAAGAAACCAAAACAATGAATTTCTGAGTTGGAAGAAGAGTTAGATCATTTAACTTTCTCATATTTAAATTAAAAAAACAAAACTCTAAAAATTTAAGTAACTTTAAGATCACATAGTTACTTAGTAGAAAAGAGTAATACCCAGCAAGCAAACTTTACAATAGATCCTTTTAAATAAGGTCCTAGGAAATATCATTCATGCCAGCATCAAAAAACTAACACTAATAATGCAAGATATTATATATTCTGCTTTTCTTACTGTCAATGAGAAAAACTATCATTCAATAAATTGCAAACCCAACACACTTAAATAAAAATAAAATGTTACTGCTAAACTAACGATAAACTACTGAATATATAGAAAGTAAGCAAACAAACTTGCCAACCTGCCAACATCTACAGATATGTTTACAGGTCAAAAATTATCAAATTATCAAGAAAGCCTGGTTCAAATTATGTATTATGTCTTTATCACAGGTCTGAAGATCAGTAAGACCTAAAACTGAAAATTATTAAACTTAAAATCTGAACAGAATATCAAATATATTTTATTCATATAAATAAAAGAATACATTACAATATTCTAAGCAAAGCAGTCTCTACTTTTGGCCTTGCTCTGTTTTCCGACCAATGTCTGCTTTTTTGCCTTGCTTTATTTTTTTATCTTATTAAATAATGTCCCTGATTAAATATTTTGAGAACAGGTAATCTGTACAATCTGAATAACACTGTTTATCTAAATATCAAACACCGTTATAACATTATGAACTGAAAGACAAACTGTACTTCTGACATCCTTACTCAGATTTCCCCTAATTGTATATTCAGTATCATTTTAAAAAACAGATTTATATTCTTTTATCAGCTCAGAAGGACTAGTATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGTAAGACACTTTGGTGGGTTTCCTTCTTGAAGCTATTATTATCAAATTCCCTATTCTTAGGACTTGTTCTAGACTAAAAGATAGTTAAGAGATATCCATCAAATACAATGTATCAACCTAAACTGGATGCTGGGGTTCTTTTTACACCCTATAAAAGACATACCTAAGACAATCAGAGAAATACAAATATGGACTTGATTATTAGATAATATAGAAGGTTTATTAATTTTCTTAGATGTGATCATGGTATTGCAGTTTTAAAGGAGAACAATCTCCTGTTTAAGAGATACATGCTGAAATATTTACGGAGTTAAAGGTCACTGGACTCCAGACTGGTGATAGAACAAGACTCTGTCTCTAAAAAATAATTAATTTTTTAAAAGAAAATAGTTTGGTAAGATGATTCTTACATTCTTAAATAACACGCCATCTAAGAAAAATGCTTTAACATAAACATTACTGAAAAAATGCTACATTTGCCACAACTTCATAAAATGTCAAGTGAAATCTCAAGCTCCAAAGATATTATTCCTATTACTAAATCTGATGTAATAACATTTTATTGATTCTAGGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAAAACCTGTCTAAGGAGAATAGACAGTAGTTAGTTCAACTTACTCATTACGTATTAGGAAGATTAACCTGGTTATCATTGTTTTATACATATATATATGAAATATATATGAGTATTCGTATAAATATAATACTTTTACCTTGTTTATGTATTTACTCAATATTCTCCTTTTCCTCTAAAATAATCTGAAGTGACTATTATCAATAAGTTTACTATGCCAAAATTCATTAATTGCCTTTCACTTAACTTTTGGGACCATAATAAATAATAAAATGTATTGCCATAACATTAATAAACTACCTTACAAAACCACCAATTAAAATCAAACAAACAAAAAAGTGTTATTTACATCTGTCAACATAAATCTACTAAAAATACATGATTTCATTCATTATATTCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGTAAGGGGACTACATTCAATCATTCATTCACTTGCTAATCTACAAATATTTACTGAGAACCTCTTATGGACCAGGTATTAGGAAAAGTAGTAACGAACGAGAAGCAGTCTCAGCCTTCATATAATTTATTATCAAACAATTACACATTTGTTAGTAAATTACACTTATTACAACTGTTATTATTTGAATTATATTTATCACAATTACATGTCTGTTCTTAAATATACTTATCACAATTTAATTCCACGGCTTACAATGATCATAACTATAATTATTAAAGACAATTTTGATTAAATGTTATGTCATAAGTAGTAACTGTTACAAATAAGCTGTGAAAAGAACCACTCCTAGCATTAGTCACTCTATTCTCTCATTAACGTTTTACATATCAATTAATTGGAAGTTAAAAGGACCAGGAAACTCAGACATACAGTATACATTTTAAAATTTCAATTATTTAAATATAATATATAGAATGTATGGCTTATAATGAATTAGTTAACTCAATGCAAATTATTCTATTTTGATTACAAATAGTAAAATAAGCAAGATAAAATAACAGATGTTTAAAATCCAAAAAGCACATACAAAAATCCATGAATGATGTCTAAGTACTCACTTATAAAGTAGAAGACATTCATTATTATATCAAATTTTTAAATGCTCAGTACTATTTGACCATTTAAAAATTTTGTATTCAAACTACCAGTGAAAGCCCTACCTAGAAGGTATACTCAGTGATAAGTTTTGTAGCTCCAAATCTTCTAATAGTGAGTGTAACCCCAAAATAAAAGGCTGACAGGTAAGTCGAGAATACTCACTTAATTCTGGTAAGAAAGCAACCCATTTGTACTTGTATTTACCAGCAATCCTTAAAATGAAGCTTCCTACTAACTCAATAGCAATAAGACAATAGTGAATGTTTAATGAAAACAGTATTTTATAAATACTTTAATAAAAAGGATTGTGATGAAGAACAATCTATTTATATTTGTTATTTGTTTTTAATTCCAATAAAAATAATTTTTAAAATTACAGAAAAAAGTTATTAAGAACCATGCTTTTAAATTTAAAATGATTTTTTAAATTTATTCCTGTCTTTTTCTACAAAGAAAGCATACATTAAGCAAATACCAAAGGCCAGGTTTACATTTGAAGAAAGTGACATTATTATTACTCAAGTCTCTAGGAATACTTAACACATCTCTTGACTGTATATGGATGTTAATAAATAGCTGACAGTAAAGTTTATCCATATAAAGACTTGCAAATATTCCTCTACCAATGACGAGACTTTAAAATATCTATAATAATGTAACACATTTCACTGGTGAAACATGTCTTGTCATATGCATTATAGAAAGGATAATCAGACTTTCAGTTATATTAATATTTTTAACATTTTTGTGCACATAGCTATCTTCAATAAAATTGTTTTAAAAGGTATTATTTTAAGATACACTAAAATGATCAAGGGATTCAAGACTAAACAACTCAATTAGTTGCACCAATAAAAAACACTTAAAAAAACTGTCAGTGTCCAACCTGTACTTAATAACTCACAGATTTTTAAAACTTTTCTTTTCAGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGTATCTTTTTCTGATACCAATACTTTATTTTCATATCTTCAAAGTATCTTCCCACATTATTAGCTATTATCTGCAATGACAACTTTTAAAAATCCGAATCCCAAATAAGCGTTTTCTCTCTAGACGAAAACCTCTTAACTATAATGAAAGTGTTCATTCTAGTTCAATCAGGTATTTTACCTCTAATCTTCCTCAGATTTTCTATTTTTTGGTAGTGTATAGATTATTTATACAGATTATTTAAAATTGGGACTTATACAGATTATTTAAAACTGGGATACATGCATCTAAAACACTGTAATATTTATAAGAAAGGAAGATAAACTTACGGGGAAATACAGTAACAGTAACTACATACGAGTCTGTACCCATTAAATTGCATATCTATCTCCTTTAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAATTTAAACATTAACCTCATTCCAAGTTAATGTGGTCTAATAATCTGGTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGATTTTTTTTATCTTAAAGTCACTGTCTATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCGTTTACATTTCTCAATCAAAATTCTTATAATACTATTTGTTTTAAATTTTGTGATGTGGGAATCAATTTTAGATGGTCACAATCTAGATTATAATCAATAGGTGAACTTATTAAATAACTTTTCTAAATAAAAAATTTAGAGACTTTTATTTTAAAAGGCATCATATGAGCTAATATCACAACTTTCCCAGTTTAAAAAACTAGTACTCTTGTTAAAACTCTAAACTTGACTAAATACAGAGGACTGGTAATTGTACAGTTCTTAAATGTTGTAGTATTAATTTCAAAACTAAAAATCGTCAGCACAGAGTATGTGTAAAAATCTGTAATACAAATTTTTAAACTGATGCTTCATTTTGCTACAAAATAATTTGGAGTAAATGTTTGATATGATTTATTTATGAAACCTAATGAAGCAGAATTAAATACTGTATTAAAATAAGTTCGCTGTCTTTAAACAAATGGAGATGACTACTAAGTCACATTGACTTTAACATGAGGTATCACTATACCTTATTTGTTAAAATATATACTGTATACATTTTATATATTTTAACACTTAATACTATGAAAACAAATAATTGTAAAGGAATCTTGTCAGATTACAGTAAGAATGAACATATTTGTGGCATCGAGTTAAAGTTTATATTTCCCCTAAATATGCTGTGATTCTAATACATTCGTGTAGGTTTTCAAGTAGAAATAAACCTCGTAACAAGTTACTGAACGTTTAAACAGCCTGACAAGCATGTATATATGTTTAAAATTCAATAAACAAAGACCCAGTCCCTAAATTATAGAAATTTAAATTATTCTTGCATGTTTATCGACATCACAACAGATCCCTAAATCCCTAAATCCCTAAAGATTAGATACAAATTTTTTACCACAGTATCACTTGTCAGAATTTATTTTTAAATATGATTTTTTAAAACTGCCAGTAAGAAATTTTAAATTAAACCCATTTGTTAAAGGATATAGTGCCCAAGTTATATGGTGACCTACCTTTGTCAATACTTAGCATTATGTATTTCAAATTATCCAATATACATGTCATATATATTTTTATATGTCACATATATAAAAGATATGTATGATCTATGTGAATCCTAAGTAAATATTTTGTTCCAGAAAAGTACAAAATAATAAAGGTAAAAATAATCTATAATTTTCAGGACCACAGACTAAGCTGTCGAAATTAACGCTGATTTTTTTAGGGCCAGAATACCAAAATGGCTCCTCTCTTCCCCCAAAATTGGACAATTTCAAATGCAAAATAATTCATTATTTAATATATGAGTTGCTTCCTCTATTTGGTTTCCTTAAAAAAAAAAAAAACTCTCATAGGACATGTTTCATTTTGTTCCTTTCAGGAGTAGTAAATTAGACGTTTTCCCCATATAAAGCTTTTTTCTACCAGAAAGATACTTCTGGTAGAAGAAGAGAAAGGAGCTCTTTATGGTTCACACGACTGTCTCCTGTCCTAACTACTTTGCTTAAAGTGCTCAAATTCCATCACTACTCACAGTTGTCTAATCTAAGTCTAATCCCCTTTGATCTCTCAGACTACCTTCCCTTTTATCTCTCTACTACTTAATAATAAGAATATCTTTTTTTCAAACTTGACCTTCATTTTGCTTTCACAATACTATACTCTCCATGGATTATCCCTTATCTGAATCCATCTTTATAACCCTATTCCTTTCTCATATTTAGTACTGTGGGCCAATGGACAACCTTCAATCATCTTTTCTACACTGACCCTCAGACATTCTATCTGCTCTCACGGACTCCTTTATTTACCATGAATAAAGTTCCAAAATCTACATATTCATCCCAAGTCTCTTTCCAGTTCCCCTTCTTACATTGCCTATTTGCCATTTCTCCCTTCAATACCCTATACTTCACTCAAATTCAACATACCAAAAATAAAAGGCCAGGCACGGTGGCTCACACCTGTAATCCCAGGACTTTGGGAGGCTGAGGCAGGTGGATCACCTGAGGTCAGGAGTCTGACCAGCCTGACCAATATGGTGAAACCCCGTCTCTACCTAAAATACAAAAATTAGCCAGGCGTGGTGGCATGTGCCTACAGTCCCAGCTACTCAAGAGGCTGAGACAGGAGAATCGCTTGAACCCAGGAGGCGGAGGTTGCAGTGAGCTGAGATCACACCAATGCACTGGGTGACAGAACAAGACTGACTCAAAAAAAAATAAATAACAAATTCCCCAGCCCCTTACTGCTACTGCTATCCCTTTCTACCCACCTTTCCCTCCTTTATACTCTTTCACACCATCTTCCTCACTTCTTTATATCCATTAATATGACCAGCATGTTCCCAGTCACAGAAGCCTGGAACCCGGAAGACATCTCTGGCTTTTCACTCAACTTTGTAAACTACCTCTTTTGTATCATAAGCCACCAAGTTCAATACAATCTTCTCTTGAAACGTCTCTTAATCTTATAAGCTTTCTTCCCCAAAGACTGTCTTTAACTTCAGTGCTAGATTATATAAGT AAD34156.1 human angiopoietin-related protein 3 (SEQ ID NO: 8)MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE

Lipoprotein (a) (LPA)

In some embodiments, the target gene for modification using thecompositions and methods disclosed herein is gene encoding lipoprotein a(LPA). LPA is a low-density lipoprotein variant. Genetic andepidemiologic studies have identified LPA as a risk factor foratherosclerosis and related diseases, such as coronary heart disease andstroke. LPA concentrations vary more than one thousand times betweenindividuals: from <0.2 to >200 mg/dL. This range of concentrations isobserved in all populations studied by scientists so far. The mean andmedian concentrations between different world populations show distinctparticularities, the main being the two to threefold higher LPA plasmaconcentration of populations of African descent compared to Asian,Oceanic, or European populations. High LPA in blood correlates withcoronary heart disease (CHD), cardiovascular disease (CVD),atherosclerosis, thrombosis, and stroke. Individuals without LPA or withvery low LPA levels seem to be healthy. Thus, plasma LPA is not vital,at least under normal environmental conditions. Since apo(a)/LPAappeared rather recently in mammalian evolution—only old world monkeysand humans have been shown to harbor LPA—its function might not bevital, but just evolutionarily advantageous under certain environmentalconditions, e.g. in case of exposure to certain infectious diseases.

An exemplary LPA amino acid sequence encoded by Human reference sequenceNG_016147.1 is provided below:

>sp|P08519|APOA_HUMAN Apolipoprotein(a) OS = Homo sapiensOX = 9606 GN = LPA PE = 1 SV = 1 (SEQ ID NO: 64)MEHKEVVLLLLLFLKSAAPEQSHVVQDCYHGDGQSYRGTYSTTVTGRTCQAWSSMTPHQHNRTTENYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDAVAAPYCYTRDPGVRWEYCNLTQCSDAEGTAVAPPTVTPVPSLEAPSEQAPTEQRPGVQECYHGNGQSYRGTYSTTVTGRTCQAWSSMTPHSHSRTPEYYPNAGLIMNYCRNPDPVAAPYCYTRDPSVRWEYCNLTQCSDAEGTAVAPPTITPIPSLEAPSEQAPTEQRPGVQECYHGNGQSYQGTYFITVTGRTCQAWSSMTPHSHSRTPAYYPNAGLIKNYCRNPDPVAAPWCYTTDPSVRWEYCNLTRCSDAEWTAFVPPNVILAPSLEAFFEQALTEETPGVQDCYYHYGQSYRGTYSTTVTGRTCQAWSSMTPHQHSRTPENYPNAGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQCLVTESSVLATLTVVPDPSTEASSEEAPTEQSPGVQDCYHGDGQSYRGSFSTTVTGRTCQSWSSMTPHWHQRTTEYYPNGGLTRNYCRNPDAEISPWCYTMDPNVRWEYCNLTQCPVTESSVLATSTAVSEQAPTEQSPTVQDCYHGDGQSYRGSFSTTVTGRTCQSWSSMTPHWHQRTTEYYPNGGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTQCPVMESTLLTTPTVVPVPSTELPSEEAPTENSTGVQDCYRGDGQSYRGTLSTTITGRTCQSWSSMTPHWHRRIPLYYPNAGLTRNYCRNPDAEIRPWCYTMDPSVRWEYCNLTRCPVTESSVLTTPTVAPVPSTEAPSEQAPPEKSPVVQDCYHGDGRSYRGISSTTVTGRTCQSWSSMIPHWHQRTPENYPNAGLTENYCRNPDSGKQPWCYTTDPCVRWEYCNLTQCSETESGVLETPTVVPVPSMEAHSEAAPTEQTPVVRQCYHGNGQSYRGTFSTTVTGRTCQSWSSMTPHRHQRTPENYPNDGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLTRCSDTEGTVVAPPTVIQVPSLGPPSEQDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCASSSFDCGKPQVEPKKCPGSIVGGCVAHPHSWPWQVSLRTRFGKHFCGGTLISPEWVLTAAHCLKKSSRPSSYKVILGAHQEVNLESHVQEIEVSRLFLEPTQADIALLKLSRPAVITDKVMPACLPSPDYMVTARTECYITGWGETQGTFGTGLLKEAQLLVIENEVCNHYKYICAEHLARGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYARVSRFVT WIEGMMRNN 

Base Editor Protein-2RNA Complexes

In another aspect, provided herein is a complex comprising the singleguide RNA as provided herein in complex with the base editor fusionprotein, e.g. the adenosine base editor fusion protein, wherein thecomplex comprises increased stability as compared to a complex with anunmodified single guide RNA and a base editor protein, wherein thestability is measured by half life of the complex ex vivo or in vitro.

In some embodiments, the complex comprises increased stability ascompared to a complex with an unmodified single guide RNA and a Cas9protein. In some embodiments, the complex comprises increased stabilityby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 200%, at least 300%, at least 400%, at least 500%,at least 600%, at least 700%, at least 800%, at least 900%, at least1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%,at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least10000%, at least 20000%, at least 30000%, at least 40000%, at least50000%, at least 60000%, at least 70000%, at least 80000%, at least90000%, or at least 100000% as compared to a complex with an unmodifiedsingle guide RNA and a Cas9 protein. In some embodiments, the complexcomprises increased stability by at least 2 fold, at least 3 fold, atleast 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, atleast 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, atleast 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, atleast 100 fold, at least 200 fold, at least 300 fold, at least 400 fold,at least 500 fold, at least 600 fold, at least 700 fold, at least 800fold, at least 900 fold, at least 1000 fold, at least 2000 fold, atleast 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or atleast 10000 fold as compared to a complex with an unmodified singleguide RNA and a Cas9 protein.

In some embodiments, wherein the stability of the complex is measured byhalf life of the complex. In some embodiments, wherein the stability ofthe complex is measured by half life of the complex ex vivo. In someembodiments, wherein the stability of the complex is measured by halflife of the complex in vitro.

In some embodiments, the complex comprises increased half life by atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 200%, at least 300%, at least 400%, at least 500%,at least 600%, at least 700%, at least 800%, at least 900%, at least1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%,at least 6000%, at least 7000%, at least 8000%, at least 9000%, at least10000%, at least 20000%, at least 30000%, at least 40000%, at least50000%, at least 60000%, at least 70000%, at least 80000%, at least90000%, or at least 100000% as compared to a complex with an unmodifiedsingle guide RNA and a Cas9 protein wherein half life of the complex ismeasured ex vivo. In some embodiments, the single guide RNA exhibitsincreased half life of the complex by at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, atleast 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, atleast 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, atleast 100 fold, at least 200 fold, at least 300 fold, at least 400 fold,at least 500 fold, at least 600 fold, at least 700 fold, at least 800fold, at least 900 fold, at least 1000 fold, at least 2000 fold, atleast 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or atleast 10000 fold as compared to a complex with an unmodified singleguide RNA and a Cas9 protein, wherein half life of the complex ismeasured ex vivo.

In some embodiments, the complex comprises increased half life whenmeasured in vitro by at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 200%, at least 300%, at least400%, at least 500%, at least 600%, at least 700%, at least 800%, atleast 900%, at least 1000%, at least 2000%, at least 3000%, at least4000%, at least 5000%, at least 6000%, at least 7000%, at least 8000%,at least 9000%, at least 10000%, at least 20000%, at least 30000%, atleast 40000%, at least 50000%, at least 60000%, at least 70000%, atleast 80000%, at least 90000%, or at least 100000% as compared to acomplex with an unmodified single guide RNA and a Cas9 protein, whereinhalf life of the complex is measured in vitro. In some embodiments, thesingle guide RNA exhibits increased half life of the complex by at least2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80fold, at least 90 fold, at least 100 fold, at least 200 fold, at least300 fold, at least 400 fold, at least 500 fold, at least 600 fold, atleast 700 fold, at least 800 fold, at least 900 fold, at least 1000fold, at least 2000 fold, at least 3000 fold, at least 4000 fold, atleast 5000 fold, at least 6000 fold, at least 7000 fold, at least 8000fold, at least 9000 fold, or at least 10000 fold as compared to acomplex with an unmodified single guide RNA and a Cas9 protein, whereinhalf life of the complex is measured in vitro.

In another aspect, provided herein is a cell comprising the complex asprovided herein. In some embodiments, the cell may be an in vitro cell.In some embodiments, the cell may be an ex vivo cell. In someembodiments, the cell may be an in vivo cell. In some embodiments, thecell may be an isolated cell.

Gene Modification Compositions

In another aspect, provided herein is a composition for genemodification comprising the single guide RNA as provided herein and abase editor protein or a nucleic acid sequence encoding the base editorprotein. In some embodiments, the composition further comprises a vectorthat comprises the nucleic acid sequence encoding the base editorprotein.

In some embodiments, the nucleic acid sequence may be a DNA, an RNA ormRNA, or a modified nucleic acid sequence. In some embodiments, thevector may be an expression vector. In some embodiments, the nucleicacid is operatively linked to a promoter of the vector. In someembodiments, the vector is a plasmid or a viral vector.

Therapeutics and Methods of Treatment

In some aspects, provided herein is a method for treating or preventinga condition in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of (i) aguide polynucleotide or a nucleic acid encoding same, and (ii) a baseeditor fusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same. In some embodiments, thebase editor system comprises a guide polynucleotide. In someembodiments, the base editor system comprises a nucleic acid encoding aguide polynucleotide. In some embodiments, the base editor systemcomprises a base editor fusion protein comprising a programmable DNAbinding domain and a deaminase. In some embodiments, the base editorsystem comprises a nucleic acid encoding a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase.

In some embodiments, the guide polynucleotide directs the base editorsystem to effect a nucleobase alteration in a ANGPTL3 gene in thesubject. In some embodiments, the guide polynucleotide directs the baseeditor system to effect a nucleobase alteration in a PCSK9gene in thesubject.

In some embodiments, the base alteration occurs in at least 35% of wholeliver cells in the subject as measured by next generation sequencing orSanger sequencing, thereby treating or preventing the condition in thesubject.

In some aspects, provided herein is a method for treating or preventinga condition in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of afirst composition, comprising (i) a guide polynucleotide or a nucleicacid encoding same, and (ii) a base editor fusion protein comprising aprogrammable DNA binding domain and a deaminase, or a nucleic acidencoding same, wherein the guide polynucleotide directs the base editorsystem to effect a nucleobase alteration in a PCSK9 gene in the subject,wherein the base alteration occurs in at least 35% of whole liver cellsin the subject as measured by next generation sequencing or Sangersequencing; and a second composition, comprising (i) a guidepolynucleotide or a nucleic acid encoding same, and (ii) a base editorfusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same, wherein the guidepolynucleotide directs the base editor system to effect a nucleobasealteration in a ANGPTL3 gene in the subject, wherein the base alterationoccurs in at least 35% of whole liver cells in the subject as measuredby next generation sequencing or Sanger sequencing.

In some embodiments, the first composition and the second compositionare administered sequentially. In some embodiment, the first compositionis administered for one or more doses, then the second composition isadministered for one or more doses. In some embodiments, the firstcomposition and the second composition interspersed. In someembodiments, the first composition and the second composition areadministered concurrently. In some embodiments, the first compositionand the second composition are administered for one or more doses. Insome embodiments, the first composition and the second composition areadministered over and interval of over an interval of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 hours. In some embodiments, the first compositionand the second composition are administered over and interval of over aninterval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In someembodiments, the first composition and the second composition areadministered over and interval of over an interval of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 weeks.

In some embodiments, the base alteration occurs in at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% of whole liver cells in the subject asmeasured by next generation sequencing or Sanger sequencing. In someembodiments, the base alteration occurs in at most 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%,99.8%, or 99.9% of whole liver cells in the subject as measured by nextgeneration sequencing or Sanger sequencing. In some embodiments, thebase alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%,5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%,20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%,50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%,80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of whole liver cells in thesubject as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%,1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%,1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%,1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%,or 1%-2% of whole liver cells in the subject as measured by nextgeneration sequencing or Sanger sequencing. In some embodiments, thebase alteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%,25%-65%, 30%-60%, 35%-55%, or 40%-50% of whole liver cells in thesubject as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 100% of whole livercells in the subject as measured by next generation sequencing or Sangersequencing.

In some embodiments, the base alteration occurs in hepatocytes in thesubject. In some embodiments, the base alteration occurs in at least 30%of hepatocytes in the subject as measured by next generation sequencingor Sanger sequencing. In some embodiments, the base alteration occurs inhepatocytes in the subject. In some embodiments, the base alterationoccurs in at least 35% of hepatocytes in the subject as measured by nextgeneration sequencing or Sanger sequencing. In some embodiments, thebase alteration occurs in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7% 99.8%, or 99.9%of hepatocytes in the subject as measured by next generation sequencingor Sanger sequencing. In some embodiments, the base alteration occurs inat most 1%, 2%, 3%, 4%, 5%0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% of hepatocytes in the subject as measuredby next generation sequencing or Sanger sequencing. In some embodiments,the base alteration occurs in 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%,5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 100%-99.9%, 15%-99.9%,20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%,50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%,80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% of hepatocytes in thesubject as measured by next generation sequencing or Sanger sequencing.In some embodiments, the base alteration occurs in 1%-99.5%, 1%-99%,1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%,1%-65%, 1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%,1%-20%, 1%-15%, 1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%,or 1%-2% hepatocytes in the subject as measured by next generationsequencing or Sanger sequencing. In some embodiments, the basealteration occurs in 1%-90%, 5%-85%, 10%-80%, 15%-75%, 20%-70%, 25%-65%,30%-60%, 35%-55%, or 40%-50% of hepatocytes in the subject as measuredby next generation sequencing or Sanger sequencing. In some embodiments,the base alteration occurs in 100% of hepatocytes in the subject asmeasured by next generation sequencing or Sanger sequencing.

In some embodiments, the base alteration occurred in whole liver cellsin the subject is measured by next generation sequencing. In someembodiments, the base alteration occurred in whole liver cells in thesubject is measured by Sanger sequencing. In some embodiments, the basealteration occurred in hepatocytes in the subject is measured by nextgeneration sequencing. In some embodiments, the base alteration occurredin hepatocytes in the subject is measured by Sanger sequencing.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administering(i) the guide polynucleotide and (ii) the nucleic acid encoding the baseeditor fusion protein to the subject. In some embodiments, the baseeditor system comprises a guide polynucleotide. In some embodiments, thebase editor system comprises a nucleic acid encoding a guidepolynucleotide.

In some embodiments, the nucleic acid encoding the base editor fusionprotein is a mRNA. In some embodiments, the mRNA generates the baseeditor fusion protein upon translation in the subject after theadministration. In some embodiments, the base editor fusion proteinforms a RNP complex in the subject.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein further comprisesadministering a lipid nanoparticle (LNP) enclosing a guidepolynucleotide or a nucleic acid encoding the guide polynucleotide (i).In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein further comprisesadministering a second LNP enclosing a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same (ii). In some embodiments, the method fortreating or preventing a condition in a subject in need thereof asdescribed herein further comprises administering a LNP enclosing a guidepolynucleotide or a nucleic acid encoding the guide polynucleotide (i)and a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same (ii). In someembodiments, the RNP complex is formed upon uptake of the LNP or thesecond LNP by a liver cell in the subject.

In some embodiments, the ratio of a guide polynucleotide or a nucleicacid encoding the guide polynucleotide (i) and a base editor fusionprotein comprising a programmable DNA binding domain and a deaminase, ora nucleic acid encoding same (ii) is about 1:10 to about 10:1 by weight.In some embodiments, the ratio of the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 1:1,1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. In someembodiments, the molar ratio of the guide polynucleotide and the nucleicacid encoding the base editor fusion protein is about 500:1 to about1:500.

In some embodiments, the ratio of the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 1000:1 toabout 1:1000 by weight. In some embodiments, the ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1,650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1,100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1,40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1,12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1,1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1,0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 by weight. In someembodiments, the ratio of the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3,1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19,1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75,1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400,1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900,1:950, or 1:1000 by weight. In some embodiments, the ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is at least about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1,700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1,200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1,50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 17:1, 15:1,14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2;1,1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1.0:1,0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 byweight. In some embodiments, the ratio of the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is at leastabout 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9,1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50,1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:150,1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650,1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or 1:1000 by weight. In someembodiments, the ratio of the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is at most about 1000:1, 950:1,900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1,400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1,70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1,18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5,1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25,1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85,1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450,1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or1:1000 by weight.

In some embodiments, the molar ratio of the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 10000:1 toabout 1:10000. In some embodiments, the molar ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1,7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1,2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1,700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1,200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1,100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1,64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1,52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1,40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1,28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1,16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1,or 0.1:1. In some embodiments, the molar ratio of the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is at least about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000,1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500,1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800,1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300,1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120,1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67,1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55,1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43,1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31,1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19,1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7,1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4,1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio of theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at least about 10000:1, 9500:1, 9000:1, 8500:1,8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1,3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1,800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1,300:1, 250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1,120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1,67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1,55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1,43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1,31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1,19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1,0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio ofthe guide polynucleotide and the nucleic acid encoding the base editorfusion protein is at least about 1:10000, 1:9500, 1:9000, 1:8500,1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000,1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850,1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350,1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130,1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68,1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56,1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44,1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32,1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20,1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5,1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the molar ratio ofthe guide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1,7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1,3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1,750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1,250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1,110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1,66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1,54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1,42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1,30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 1:10000, 1:9500, 1:9000, 1:8500, 1:8000,1:7500, 1:7000, 1:6500, 1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500,1:3000, 1:2500, 1:2000, 1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800,1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300,1:250, 1:200, 1:190, 1:180, 1:170, 1:160, 1:150, 1:140, 1:130, 1:120,1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67,1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55,1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43,1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31,1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19,1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7,1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4,1:0.3, 1:0.2, or 1:0.1.

In some embodiments, the ratio of a nucleic acid encoding the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 10:1 to about 1:10 by weight. In some embodiments, theratio of a nucleic acid encoding the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 4:1, 3;1,2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, or 1:4 by weight. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is about 500:1to about 1:500.

In some embodiments, the ratio of a nucleic acid encoding the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is about 1000:1 to about 1:1000 by weight. In some embodiments,the ratio of a nucleic acid encoding the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is about 1000:1,950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1,450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1,75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1,19:1, 18:1, 17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1 by weight. In some embodiments, the ratio of anucleic acid encoding the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is about 1:0.1, 1:0.2, 1:0.3,1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3,1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19,1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75,1:80, 1:85, 1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400,1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900,1:950, or 1:1000 by weight. In some embodiments, the ratio of a nucleicacid encoding the guide polynucleotide and the nucleic acid encoding thebase editor fusion protein is at least about 1000:1, 950:1, 900:1,850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1,350:1, 300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1,65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1,17:1, 17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3: 1, 1.2:1,1.1:1, 1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or0.1 by weight. In some embodiments, the ratio of a nucleic acid encodingthe guide polynucleotide and the nucleic acid encoding the base editorfusion protein is at least about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5,1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25,1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85,1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450,1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or1:1000 by weight. In some embodiments, the ratio of a nucleic acidencoding the guide polynucleotide and the nucleic acid encoding the baseeditor fusion protein is at most about 1000:1, 950:1, 900:1, 850:1,800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1,300:1, 250:1, 200:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1,60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1,17:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1, 2;1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1,1.0:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 byweight. In some embodiments, the ratio of a nucleic acid encoding theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is at most about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5,1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25,1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85,1:90, 1:95, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450,1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, or1:1000 by weight.

In some embodiments, the molar ratio of a nucleic acid encoding theguide polynucleotide and the nucleic acid encoding the base editorfusion protein is about 10000:1 to about 1:10000. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is about10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1,5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1,1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1,500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1,160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1,75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1,59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1,47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1,35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1,23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1,11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1,0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is at leastabout 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500,1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000,1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600,1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180,1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90,1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62,1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50,1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38,1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26,1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14,1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1,1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. Insome embodiments, the molar ratio of a nucleic acid encoding the guidepolynucleotide and the nucleic acid encoding the base editor fusionprotein is at least about 10000:1, 9500:1, 9000:1, 8500:1, 8000:1,7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1,3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1,750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1,250:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1,110:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 69:1, 68:1, 67:1,66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1, 59:1, 58:1, 57:1, 56:1, 55:1,54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1, 47:1, 46:1, 45:1, 44:1, 43:1,42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1, 35:1, 34:1, 33:1, 32:1, 31:1,30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1,0.3:1, 0.2:1, or 0.1:1. In some embodiments, the molar ratio of anucleic acid encoding the guide polynucleotide and the nucleic acidencoding the base editor fusion protein is at least about 1:10000,1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500, 1:6000, 1:5500,1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000, 1:1500, 1:1000,1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500,1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180, 1:170, 1:160,1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75,1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62, 1:61, 1:60, 1:59,1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50, 1:49, 1:48, 1:47,1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38, 1:37, 1:36, 1:35,1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26, 1:25, 1:24, 1:23,1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11,1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7,1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, themolar ratio of a nucleic acid encoding the guide polynucleotide and thenucleic acid encoding the base editor fusion protein is at most about10000:1, 9500:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1,5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1,1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1,500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 190:1, 180:1, 170:1,160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, 95:1, 90:1, 85:1, 80:1,75:1, 70:1, 69:1, 68:1, 67:1, 66:1, 65:1, 64:1, 63:1, 62:1, 61:1, 60:1,59:1, 58:1, 57:1, 56:1, 55:1, 54:1, 53:1, 52:1, 51:1, 50:1, 49:1, 48:1,47:1, 46:1, 45:1, 44:1, 43:1, 42:1, 41:1, 40:1, 39:1, 38:1, 37:1, 36:1,35:1, 34:1, 33:1, 32:1, 31:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1,23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1,11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1,0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1. In some embodiments,the molar ratio of a nucleic acid encoding the guide polynucleotide andthe nucleic acid encoding the base editor fusion protein is at mostabout 1:10000, 1:9500, 1:9000, 1:8500, 1:8000, 1:7500, 1:7000, 1:6500,1:6000, 1:5500, 1:5000, 1:4500, 1:4000, 1:3500, 1:3000, 1:2500, 1:2000,1:1500, 1:1000, 1:950, 1:900, 1:850, 1:800, 1:750, 1:700, 1:650, 1:600,1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:190, 1:180,1:170, 1:160, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:95, 1:90,1:85, 1:80, 1:75, 1:70, 1:69, 1:68, 1:67, 1:66, 1:65, 1:64, 1:63, 1:62,1:61, 1:60, 1:59, 1:58, 1:57, 1:56, 1:55, 1:54, 1:53, 1:52, 1:51, 1:50,1:49, 1:48, 1:47, 1:46, 1:45, 1:44, 1:43, 1:42, 1:41, 1:40, 1:39, 1:38,1:37, 1:36, 1:35, 1:34, 1:33, 1:32, 1:31, 1:30, 1:29, 1:28, 1:27, 1:26,1:25, 1:24, 1:23, 1:22, 1:21, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1: 14,1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1,1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1.

In some embodiments, the nucleobase alteration results in a reduction ofat least 35% in blood PCSK9 protein level in the subject as compared toprior to the administration as measured by ELISA, Western blots, orLC-MS/MS.

In some embodiments, the nucleobase alteration results in a reduction ofat least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% in blood PCSK9 protein level in the subjectas compared to prior to the administration as measured by ELISA, Westernblots, or LC-MS/MS.

In some embodiments, the nucleobase alteration results in a reduction ofat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%,95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or99.9% in blood PCSK9 protein level in the subject as compared to priorto the administration as measured by ELISA, Western blots, or LC-MS/MS.In some embodiments, the nucleobase alteration results in a reduction ofat most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%,95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or99.9% in blood PCSK9 protein level in the subject as compared to priorto the administration as measured by ELISA, Western blots, or LC-MS/MS.In some embodiments, the nucleobase alteration results in a reduction of1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%,8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%,30%-99.9%, 31%-99.9%, 32%-99.9%, 33%-99.9%, 34%-99.9%, 35%-99.9%,36%-99.9%, 37%-99.9%, 38%-99.9%, 39%-99.9%, 40%-99.9%, 45%-99.9%,50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%,80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood PCSK9 proteinlevel in the subject as compared to prior to the administration asmeasured by ELISA, Western blots, or LC-MS/MS. In some embodiments, thenucleobase alteration results in a reduction of 1%-99.5%, 1%-99%,1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-79%, 1%-78%,1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%, 1%-72%, 1%-71%, 1%-70%, 1%-65%,1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%,1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%,1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% inblood PCSK9 protein level in the subject as compared to prior to theadministration as measured by ELISA, Western blots, or LC-MS/MS. In someembodiments, the nucleobase alteration results in a reduction of1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%,31%-80%, 32%-79%, 33%-78%, 34%-77%, 35%-76%, 36%-76%, 37%-75%, 38%-74%,39%-73%, 40%-72%, 45%-71%, 50%-70%, or 55%-65% in blood PCSK9 proteinlevel in the subject as compared to prior to the administration asmeasured by ELISA, Western blots, or LC-MS/MS. In some embodiments, thenucleobase alteration results in a reduction of 100% in blood PCSK9protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS.

In some embodiments, the nucleobase alteration results in at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%,290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood PCSK9protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS. In some embodiments,the nucleobase alteration results in at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood PCSK9protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS.

In some embodiments, the reduction of blood PCSK9 protein level or theblood PCSK9 protein level in the subject as compared to prior to theadministration is measured by ELISA (enzyme-linked immunosorbent assay).In some embodiments, the reduction of blood PCSK9 protein level or theblood PCSK9 protein level in the subject as compared to prior to theadministration is measured by Western blot analysis. In someembodiments, the reduction of blood PCSK9 protein level or the bloodPCSK9 protein level in the subject as compared to prior to theadministration is measured by LC-MS/MS (liquid chromatography-tandemmass spectrometry).

In some embodiments, the nucleobase alteration results in a reduction ofat least 35% in blood ANGPTL3 protein level in the subject as comparedto prior to the administration as measured by ELISA, Western blots, orLC-MS/MS.

In some embodiments, the nucleobase alteration results in a reduction ofat least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% in blood ANGPTL3 protein level in the subjectas compared to prior to the administration as measured by ELISA, Westernblots, or LC-MS/MS.

In some embodiments, the nucleobase alteration results in a reduction ofat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%,95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or99.9% in blood ANGPTL3 protein level in the subject as compared to priorto the administration as measured by ELISA, Western blots, or LC-MS/MS.In some embodiments, the nucleobase alteration results in a reduction ofat most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%,95%, 97%, 98%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or99.9% in blood ANGPTL3 protein level in the subject as compared to priorto the administration as measured by ELISA, Western blots, or LC-MS/MS.In some embodiments, the nucleobase alteration results in a reduction of1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%,8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%,30%-99.9%, 31%-99.9%, 32%-99.9%, 33%-99.9%, 34%-99.9%, 35%-99.9%,36%-99.9%, 37%-99.9%, 38%-99.9%, 39%-99.9%, 40%-99.9%, 45%-99.9%,50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%,80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% in blood ANGPTL3 proteinlevel in the subject as compared to prior to the administration asmeasured by ELISA, Western blots, or LC-MS/MS. In some embodiments, thenucleobase alteration results in a reduction of 1%-99.5%, 1%-99%,1%-98%, 1%-97%, 1%-96%, 1%-95%, 1%-90%, 1%-85%, 1%-80%, 1%-79%, 1%-78%,1%-77%, 1%-76%, 1%-75%, 1%-74%, 1%-73%, 1%-72%, 1%-71%, 1%-70%, 1%-65%,1%-60%, 1%-55%, 1%-50%, 1%-45%, 1%-40%, 1%-39%, 1%-38%, 1%-37%, 1%-36%,1%-35%, 1%-34%, 1%-33%, 1%-32%, 1%-31%, 1%-30%, 1%-25%, 1%-20%, 1%-15%,1%-10%, 1%-9%, 1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% inblood ANGPTL3 protein level in the subject as compared to prior to theadministration as measured by ELISA, Western blots, or LC-MS/MS. In someembodiments, the nucleobase alteration results in a reduction of1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%,31%-80%, 32%-79%, 33%-78%, 34%-77%, 35%-76%, 36%-76%, 37%-75%, 38%-74%,39%-73%, 40%-72%, 45%-71%, 50%-70%, or 55%-65% in blood ANGPTL3 proteinlevel in the subject as compared to prior to the administration asmeasured by ELISA, Western blots, or LC-MS/MS. In some embodiments, thenucleobase alteration results in a reduction of 100% in blood ANGPTL3protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS.

In some embodiments, the nucleobase alteration results in at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%,290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000% less blood ANGPTL3protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS. In some embodiments,the nucleobase alteration results in at least 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, or more than 10-fold less blood ANGPTL3protein level in the subject as compared to prior to the administrationas measured by ELISA, Western blots, or LC-MS/MS.

In some embodiments, the reduction of blood ANGPTL3 protein level or theblood ANGPTL3 protein level in the subject as compared to prior to theadministration is measured by ELISA (enzyme-linked immunosorbent assay).In some embodiments, the reduction of blood ANGPTL3 protein level or theblood ANGPTL3 protein level in the subject as compared to prior to theadministration is measured by Western blot analysis. In someembodiments, the reduction of blood ANGPTL3 protein level or the bloodANGPTL3 protein level in the subject as compared to prior to theadministration is measured by LC-MS/MS (liquid chromatography-tandemmass spectrometry).

In some embodiments, the nucleobase alteration results in a reduction ofat least 35% in blood low-density lipoprotein cholesterol (LDL-C) levelin the subject as compared to prior to the administration.

In some embodiments, the nucleobase alteration results in a reduction ofat least 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood low-density lipoproteincholesterol (LDL-C) level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%, 99.5%, 99.7%, 99.8%, or 99.9%in blood low-density lipoprotein cholesterol (LDL-C) level in thesubject as compared to prior to the administration. In some embodiments,the nucleobase alteration results in a reduction of at most 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% in blood low-density lipoproteincholesterol (LDL-C) level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of 1%-99.9%, 2%-99.9%, 3%-99.9%, 4%-99.9%, 5%-99.9%,6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%, 10%-99.9%, 15%-99.9%, 20%-99.9%,25%-99.9%, 30%-99.9%, 35%-99.9%, 40%-99.9%, 45%-99.9%, 50%-99.9%,55%-99.9%, 60%-99.9%, 65%-99.9%, 70%-99.9%, 75%-99.9%, 80%-99.9%,85%-99.9%, 90%-99.9%, or 95-99.9% in blood low-density lipoproteincholesterol (LDL-C) level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%,1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%,1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%,1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood low-densitylipoprotein cholesterol (LDL-C) level in the subject as compared toprior to the administration. In some embodiments, the nucleobasealteration results in a reduction of 1%-99.9%, 5%-99.5%, 10%-99%,15%-97%, 20%-95%, 25%-90%, 30%-85%, 35%-80%, 40%-75%, 45%-70%, 50%-65%,or 55%-60% in blood low-density lipoprotein cholesterol (LDL-C) level inthe subject as compared to prior to the administration. In someembodiments, the nucleobase alteration results in a reduction of 100% inblood low-density lipoprotein cholesterol (LDL-C) level in the subjectas compared to prior to the administration. In some embodiments, thenucleobase alteration results in at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 1%, 2%, %13%, %14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%,90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 400% 500%,600%, 700%, 800%, 900%, 1000% less blood low-density lipoproteincholesterol (LDL-C) level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, ormore than 10-fold less blood low-density lipoprotein cholesterol (LDL-C)level in the subject as compared to prior to the administration.

In some embodiments, the nucleobase alteration results in a reduction ofat least 35% in blood triglyceride level in the subject as compared toprior to the administration.

In some embodiments, the nucleobase alteration results in a reduction ofat least 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% in blood triglyceride level in thesubject as compared to prior to the administration. In some embodiments,the nucleobase alteration results in a reduction of at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%,99.3%, 99.5%, 99.7%, 99.8%, or 99.9% in blood triglyceride level in thesubject as compared to prior to the administration. In some embodiments,the nucleobase alteration results in a reduction of at most 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.3%,99.5%, 99.7%, 99.8%, or 99.9% in blood triglyceride level in the subjectas compared to prior to the administration. In some embodiments, thenucleobase alteration results in a reduction of 1%-99.9%, 2%-99.9%,3%-99.9%, 4%-99.9%, 5%-99.9%, 6%-99.9%, 7%-99.9%, 8%-99.9%, 9%-99.9%,10%-99.9%, 15%-99.9%, 20%-99.9%, 25%-99.9%, 30%-99.9%, 35%-99.9%,40%-99.9%, 45%-99.9%, 50%-99.9%, 55%-99.9%, 60%-99.9%, 65%-99.9%,70%-99.9%, 75%-99.9%, 80%-99.9%, 85%-99.9%, 90%-99.9%, or 95-99.9% inblood triglyceride level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin a reduction of 1%-99.5%, 1%-99%, 1%-98%, 1%-97%, 1%-96%, 1%-95%,1%-90%, 1%-85%, 1%-80%, 1%-75%, 1%-70%, 1%-65%, 1%-60%, 1%-55%, 1%-50%,1%-45%, 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-9%,1%-8%, 1%-7%, 1%-6%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% in blood triglyceridelevel in the subject as compared to prior to the administration. In someembodiments, the nucleobase alteration results in a reduction of1%-99.9%, 5%-99.5%, 10%-99%, 15%-97%, 20%-95%, 25%-90%, 30%-85%,35%-80%, 40%-75%, 45%-70%, 50%-65%, or 55%-60% in blood triglyceridelevel in the subject as compared to prior to the administration. In someembodiments, the nucleobase alteration results in a reduction of 100% inblood triglyceride level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%,260%, 270%, 280%, 290%, 300%, 400% 500%, 600%, 700%, 800%, 900%, 1000%less blood triglyceride level in the subject as compared to prior to theadministration. In some embodiments, the nucleobase alteration resultsin at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, ormore than 10-fold less blood triglyceride level in the subject ascompared to prior to the administration.

In some embodiments, the blood triglyceride level or the reduction ofblood triglyceride level in the subject as compared to prior to theadministration is measured by any standard technique. In someembodiments, the blood low-density lipoprotein cholesterol (LDL-C) levelor the reduction of blood low-density lipoprotein cholesterol (LDL-C)level in the subject as compared to prior to the administration ismeasured by any standard technique. For example, a clinical analyzerinstrument may be used to measure a ‘lipid panel’ in serum samples whichentails the direct measurement of cholesterol (total C), triglycerides(TG) and high-density lipoprotein cholesterol (HDL-C) enzymatically.Reagent kits specific for each analyte contain buffers, calibrators,blanks and controls. As used in the present disclosure, cholesterol,triglycerides and HDL-C may be quantified using absorbance measurementsof specific enzymatic reaction products. LDL-C may be determinedindirectly. In some instances, most of circulating cholesterol can befound in three major lipoprotein fractions: very low-densitylipoproteins (VLDL), LDL and HDL. In some embodiments, total circulatingcholesterol may be estimated with the formula [TotalC]=[VLDL-C]+[LDL-C]+[HDL-C]. Thus the LDL-C can be calculated frommeasured values of total cholesterol, triglycerides and HDL-C accordingto the relationship: [LDL-C]=[total C]− [HDL-C]-[TG]/5, where [TG]/5 isan estimate of VLDL-cholesterol. A reagent kit specific fortriglycerides containing buffers, calibrators, blanks and controls. Asused herein, serum samples from the study may be analyzed andtriglycerides may be measured using a series of coupled enzymaticreactions. In some embodiments, H₂O₂ may be used to quantify theanalyte.as the end product of the last one and its absorbance at 500 nm,and the color intensity is proportional to triglyceride concentrations.

In some embodiments, the guide polynucleotide is a guide RNA. In someembodiments, the guide RNA comprises a spacer sequence that binds to thecomplementary strand of a protospacer sequence of the ANGPTL3 gene with0, 1, or 2 mismatches. In some embodiments, the guide RNA comprises aspacer sequence that binds to the complementary strand of a protospacersequence of the ANGPTL3 gene with no mismatches. In some embodiments,the guide RNA comprises a spacer sequence that binds to thecomplementary strand of a protospacer sequence of the ANGPTL3 gene with1 mismatch. In some embodiments, the guide RNA comprises a spacersequence that binds to the complementary strand of a protospacersequence of the ANGPTL3 gene with 2 mismatches. In some embodiments, theguide RNA comprises a spacer sequence that binds to the complementarystrand of a protospacer sequence of the ANGPTL3 gene with 3 mismatches.In some embodiments, the guide RNA comprises a spacer sequence thatbinds to the complementary strand of a protospacer sequence of theANGPTL3 gene with 4 mismatches. In some embodiments, the guide RNAcomprises a spacer sequence that binds to the complementary strand of aprotospacer sequence of the ANGPTL3 gene with 5 mismatches.

In some embodiments, the guide polynucleotide is a guide RNA. In someembodiments, the guide RNA comprises a spacer sequence that binds to thecomplementary strand of a protospacer sequence of the PCSK9 gene with 0,1, or 2 mismatches. In some embodiments, the guide RNA comprises aspacer sequence that binds to the complementary strand of a protospacersequence of the PCSK9 gene with no mismatches. In some embodiments, theguide RNA comprises a spacer sequence that binds to the complementarystrand of a protospacer sequence of the PCSK9 gene with 1 mismatch. Insome embodiments, the guide RNA comprises a spacer sequence that bindsto the complementary strand of a protospacer sequence of the PCSK9 genewith 2 mismatches. In some embodiments, the guide RNA comprises a spacersequence that binds to the complementary strand of a protospacersequence of the PCSK9 gene with 3 mismatches. In some embodiments, theguide RNA comprises a spacer sequence that binds to the complementarystrand of a protospacer sequence of the PCSK9 gene with 4 mismatches. Insome embodiments, the guide RNA comprises a spacer sequence that bindsto the complementary strand of a protospacer sequence of the PCSK9 genewith 5 mismatches.

In some embodiments, the nucleobase alteration is outside of theprotospacer sequence in less than 1% of whole liver cells in the subjectas measured by net nucleobase editing. In some embodiments, thenucleobase alteration is outside of the protospacer sequence in lessthan 1% of hepatocytes in the subject as measured by net nucleobaseediting. In some embodiments, the nucleobase alteration is only withinthe protospacer sequence as measured by net nucleobase editing.

In some embodiments, the nucleobase alteration is outside of theprotospacer sequence in less than 0.01%. 0.02%, 0.03% 0.04%, 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%0, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%0, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 65%, 80%,85%, 90% of whole liver cells in the subject as measured by netnucleobase editing. In some embodiments, the nucleobase alteration isoutside of the protospacer sequence in less than 0.01%. 0.02%, 0.03%0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%,9.0%0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,65%, 80%, 85%, 90% of hepatocytes in the subject as measured by netnucleobase editing. In some embodiments, the nucleobase alteration isoutside of the protospacer sequence in less than 0.01%. 0.02%, 0.03%0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0% 4.0%, 5.0%, 6.0%, 7.0%, 8.0%,9.0%0, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,65%, 80%, 85%, 90% of cells in the subject as measured by net nucleobaseediting.

In some embodiments, the administration is via intravenous infusion. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises sequentialadministration of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding the guide polynucleotide and the second LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding same. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises concurrentadministration of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding the guide polynucleotide and the second LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding same. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same followed by staggered doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding theguide polynucleotide over an interval of 1, 2, 3, 4, 5, 6, or 7 days.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same followed by staggered doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding theguide polynucleotide over an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 hours. In some embodiments, the method for treating orpreventing a condition in a subject in need thereof as described hereincomprises administering a single dose of the LNP enclosing (ii) a baseeditor fusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same followed by staggered dosesof the LNP enclosing (i) a guide polynucleotide or a nucleic acidencoding the guide polynucleotide over an interval of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 days. In some embodiments, the method for treatingor preventing a condition in a subject in need thereof as describedherein comprises administering a single dose of the LNP enclosing (ii) abase editor fusion protein comprising a programmable DNA binding domainand a deaminase, or a nucleic acid encoding same followed by staggereddoses of the LNP enclosing (i) a guide polynucleotide or a nucleic acidencoding the guide polynucleotide over an interval of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months.

The method as described herein may be used for treating or preventing acondition in a subject in need thereof. In some embodiments, the methodcomprises administering a single dose of a LNP enclosing (i) a guidepolynucleotide, e.g., a guide RNA, or a nucleic acid encoding the guidepolynucleotide and (ii) a base editor fusion protein or a nucleic acidencoding the base editor fusion protein, e.g., an mRNA encoding the baseeditor fusion protein. For example, the method may compriseadministering an LNP enclosing (i) a guide RNA and (i) an mRNA encodingthe base editor fusion protein. The LNP may be administered at a singledose or multiple doses. In other embodiments, the method comprisesadministering a LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding the guide polynucleotide, and a second LNP enclosing (ii)the base editor fusion protein or a nucleic acid encoding the baseeditor fusion protein. The LNP may be administered at a single dose ormultiple doses.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding same and administering multiple doses of the LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 1 day, andthe multiple doses are given at 2 day intervals. In some embodiments,the method for treating or preventing a condition in a subject in needthereof as described herein comprises administering a single dose of theLNP enclosing (i) a guide polynucleotide or a nucleic acid encoding sameand administering multiple doses of the LNP enclosing (ii) a base editorfusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same after 1 day, and the multipledoses are given at 3 day intervals. In some embodiments, the method fortreating or preventing a condition in a subject in need thereof asdescribed herein comprises administering a single dose of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding same andadministering multiple doses of the LNP enclosing (ii) a base editorfusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same after 1 day, and the multipledoses are given at 4 day intervals. In some embodiments, the method fortreating or preventing a condition in a subject in need thereof asdescribed herein comprises administering a single dose of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding same andadministering multiple doses of the LNP enclosing (ii) a base editorfusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same after 1 day, and the multipledoses are given at 5 day intervals. In some embodiments, the method fortreating or preventing a condition in a subject in need thereof asdescribed herein comprises administering a single dose of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding same andadministering multiple doses of the LNP enclosing (ii) a base editorfusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same after 1 day, and the multipledoses are given at 6 day intervals. In some embodiments, the method fortreating or preventing a condition in a subject in need thereof asdescribed herein comprises administering a single dose of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding same andadministering multiple doses of the LNP enclosing (ii) a base editorfusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same after 1 day, and the multipledoses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding same and administering multiple doses of the LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 2 days,and the multiple doses are given at 2 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 2 days,and the multiple doses are given at 3 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 2 days,and the multiple doses are given at 4 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 2 days,and the multiple doses are given at 5 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 2 days,and the multiple doses are given at 6 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 2 days,and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding same and administering multiple doses of the LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 3 days,and the multiple doses are given at 2 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 3 days,and the multiple doses are given at 3 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 3 days,and the multiple doses are given at 4 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 3 days,and the multiple doses are given at 5 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 3 days,and the multiple doses are given at 6 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 3 days,and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding same and administering multiple doses of the LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 4 days,and the multiple doses are given at 2 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 4 days,and the multiple doses are given at 3 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 4 days,and the multiple doses are given at 4 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 4 days,and the multiple doses are given at 5 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 4 days,and the multiple doses are given at 6 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 4 days,and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding same and administering multiple doses of the LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 5 days,and the multiple doses are given at 2 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 5 days,and the multiple doses are given at 3 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 5 days,and the multiple doses are given at 4 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 5 days,and the multiple doses are given at 5 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 5 days,and the multiple doses are given at 6 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 5 days,and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding same and administering multiple doses of the LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 6 days,and the multiple doses are given at 2 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 6 days,and the multiple doses are given at 3 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 6 days,and the multiple doses are given at 4 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 6 days,and the multiple doses are given at 5 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 6 days,and the multiple doses are given at 6 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 6 days,and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding same and administering multiple doses of the LNPenclosing (ii) a base editor fusion protein comprising a programmableDNA binding domain and a deaminase, or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 7 days,and the multiple doses are given at 2 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 7 days,and the multiple doses are given at 3 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 7 days,and the multiple doses are given at 4 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 7 days,and the multiple doses are given at 5 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 7 days,and the multiple doses are given at 6 day intervals. In someembodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (i) a guide polynucleotide or a nucleicacid encoding same and administering multiple doses of the LNP enclosing(ii) a base editor fusion protein comprising a programmable DNA bindingdomain and a deaminase, or a nucleic acid encoding same after 7 days,and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 2 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 3 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 4 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 5 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 6 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 1 day, and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 2 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 3 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 4 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 5 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 6 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 2 days, and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 2 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 3 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 4 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 5 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 6 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 3 days, and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 2 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 3 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 4 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 5 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 6 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 4 days, and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 2 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 3 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 4 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 5 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 6 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 5 days, and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 2 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 3 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 4 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 5 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 6 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 6 days, and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 1 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 2 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 3 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 4 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 5 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 6 day intervals. Insome embodiments, the method for treating or preventing a condition in asubject in need thereof as described herein comprises administering asingle dose of the LNP enclosing (ii) a base editor fusion proteincomprising a programmable DNA binding domain and a deaminase, or anucleic acid encoding same and administering multiple doses of the LNPenclosing (i) a guide polynucleotide or a nucleic acid encoding sameafter 7 days, and the multiple doses are given at 7 day intervals.

In some embodiments, the method for treating or preventing a conditionin a subject in need thereof as described herein comprises administeringa single dose of the LNP enclosing (i) a guide polynucleotide or anucleic acid encoding the guide polynucleotide and (ii) a base editorfusion protein comprising a programmable DNA binding domain and adeaminase, or a nucleic acid encoding same. In some embodiments, thesingle dose of the LNP is at 0.3 mg/kg. In some embodiments, the singledose of the LNP is at 0.5 mg/kg. In some embodiments, the single dose ofthe LNP is at 1 mg/kg. In some embodiments, the single dose of the LNPis at 1 mg/kg. In some embodiments, the single dose of the LNP is atabout 0.3 to about 3 mg/kg.

Methods for treatment of a condition as provided herein may comprise atreatment course of one or more treatments, with each treatmentcomprising a single dose or multiple doses of a base editor system, or aLNP enclosing one or more components of the base editor system. Forexample, a subject in need thereof may be administered a single dose ofa LNP enclosing a mRNA encoding a base editor fusion protein and a guideRNA for a treatment. In some embodiments, a subject may be administereda treatment course of one or more treatments, wherein each treatmentcomprises one or more of the single doses of the LNP, for example, asubject may also be administered 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, or more doses of a LNP enclosing a mRNA encoding a base editorfusion protein and a guide RNA for a treatment. The subject may receivea treatment course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, ormore treatments, where each dose may be administered 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2months, 3 months, 6 months, 9 months, 12 months, 24 months, 48 months orapart. A single dose for the treatment as described herein may comprisea LNP enclosing a guide RNA or a mRNA encoding the base editor fusionprotein, or both.

In some embodiments, the single dose of the LNP comprises 0.1 mg/kg, 0.2mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg,0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg,2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg,3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg,4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg, 5.4mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg, 6.0 mg/kg,6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg,7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg, 8.0mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6 mg/kg,8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg,10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110mg/kg, 115 mg/kg, 120 mg/kg, 125 mg/kg, 130 mg/kg, 135 mg/kg, 140 mg/kg,145 mg/kg, 150 mg/kg, 155 mg/kg, 160 mg/kg, 165 mg/kg, 170 mg/kg, 175mg/kg, 180 mg/kg, 185 mg/kg, 190 mg/kg, 195 mg/kg, 200 mg/kg, 250 mg/kg,300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, or 500 mg/kg.

In some embodiments, the condition is a atherosclerotic cardiovasculardisease. In some embodiments, the condition is a cardiovascular disease,or diabetes. In some embodiments, the condition is a atheroscleroticvascular disease.

In some embodiments, the subject is a non-human primate. In someembodiments, the subject is a monkey. In some embodiments, the subjectis a human. In some embodiments, the deaminase is an adenine deaminase.In some embodiments, the nucleobase alteration is a A•T to G•Calteration. In some embodiments, the deaminase is an adenine deaminaseand the nucleobase alteration is a A•T to G•C alteration. In someembodiments, the programmable DNA binding domain comprises a nucleaseinactive Cas9. In some embodiments, the programmable DNA binding domaincomprises a Cas9 nickase. In some embodiments, the programmable DNAbinding domain comprises a Cas9.

In some embodiments, the nucleobase alteration is at a splice site ofthe PCSK9 gene. In some embodiments, the nucleobase alteration is at asplice donor site of the PCSK9 gene. In some embodiments, the splicedonor site is at 5′ end of PCSK9 intron 1 as referenced in SEQ ID NO: 5.In some embodiments, the nucleobase alteration is at a splice acceptorsite of the PCSK9 gene. In some embodiments, the nucleobase alterationresults in a frame shift, a premature stop codon, a insertion ordeletion in a transcript encoded by the PCSK9 gene. In some embodiments,the nucleobase alteration results in an aberrant transcript encoded bythe PCSK9 gene. In some embodiments, the guide polynucleotide is a guideRNA. In some embodiments, the guide RNA is chemically modified. In someembodiments, the guide RNA comprises a tracrRNA sequence. In someembodiments, the guide RNA comprises a chemical modification as setforth in Table 1 or Table 24.

In some embodiments, the nucleobase alteration is at a splice site ofthe ANGPTL3 gene. In some embodiments, the nucleobase alteration is at asplice donor site of the ANGPTL3 gene. In some embodiments, the splicedonor site is at 5′ end of ANGPTL3 intron 6 as referenced in SEQ ID NO:7. In some embodiments, the nucleobase alteration is at a spliceacceptor site of the ANGPTL3 gene. In some embodiments, the nucleobasealteration results in a frame shift, a premature stop codon, a insertionor deletion in a transcript encoded by the ANGPTL3 gene. In someembodiments, the nucleobase alteration results in an aberrant transcriptencoded by the ANGPTL3 gene. In some embodiments, the guidepolynucleotide is a guide RNA. In some embodiments, the guide RNA ischemically modified. In some embodiments, the guide RNA comprises atracrRNA sequence. In some embodiments, the guide RNA comprises achemical modification as set forth in Table 1 or Table 24.

In some embodiments, the guide RNA comprises a guide RNA sequence setforth in Table 1 or Table 24. In some embodiments, the guide RNAcomprises the sequence5′-5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggCUaGUC cGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu-3′ (SEQ ID NO: 9),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUcc GUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu-3′ (SEQ ID NO: 9) (GA346),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAacAAcuugaaaaagugGcaccgagucggugcusususu-3 (SEQ ID NO: 65) (GA374),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuuu-3′ (SEQ ID NO: 11)(GA385),5′-cscscsGCACCUUGGCGCAGCGGgUUUUAGagcuagaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuUu-3′ (SEQ ID NO: 11) (GA386)or 5′-cscscsGCACCUUGGCGCAGCGgUUUUAGagcuaGaaauagcaaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcuususuuuu-3′ (SEQ ID NO: 12) (GA387).

In some embodiments, the protospacer sequence comprises a protospacersequence set forth in Table 1 or Table 24. In some embodiments, theprotospacer comprises the sequence

(SEQ ID NO: 13) 5′-CCCGCACCTTGGCGCAGCGG-3′, (SEQ ID NO: 14)AAGATACCTGAATAACTCTC-3′ or (SEQ ID NO: 15) 5′-AAGATACCTGAATAACCCTC-3′.

In some embodiments, the base editor fusion protein comprises thesequence of SEQ ID NO: 3. In some embodiments, the adenosine deaminasecomprises an amino acid sequence that is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO:3 or to any of the adenosine deaminases provided herein. It should beappreciated that adenosine deaminases provided herein may include one ormore mutations (e.g., any of the mutations provided herein). Thedisclosure provides any deaminase domains with a certain percentidentity plus any of the mutations or combinations thereof describedherein. In some embodiments, the adenosine deaminase comprises an aminoacid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,or more mutations compared to the amino acid sequence set forth in SEQID NO: 3 or any of the adenosine deaminases provided herein. In someembodiments, the adenosine deaminase comprises an amino acid sequencethat has at least 5, at least 10, at least 15, at least 20, at least 25,at least 30, at least 35, at least 40, at least 45, at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least110, at least 120, at least 130, at least 140, at least 150, at least160, or at least 170 identical contiguous amino acid residues ascompared to any one of the amino acid sequences set forth in SEQ ID NO:3 or any of the adenosine deaminases provided herein.

In some embodiments, the mRNA comprises a cap analog.

In some embodiments, the mRNA comprises at least 1, 2, or 3 nucleotidesat the 5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, oradditional 2′ chemical modification or a combination thereof. In someembodiments, the mRNA comprises at least 1, 2, or 3 nucleotides at the5′ end that comprises 2′-hydroxyl group, 2′-O-methyl group, oradditional 2′ chemical modification or a combination thereof. In someembodiments, the mRNA comprises at least 1 nucleotide at the 5′ end thatcomprises 2′-hydroxyl group, 2′-O-methyl group, or additional 2′chemical modification or a combination thereof. In some embodiments, themRNA comprises at least 2 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 3 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 4 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 5 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 6 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 7 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 8 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 9 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof. In some embodiments, the mRNAcomprises at least 10 nucleotides at the 5′ end that comprises2′-hydroxyl group, 2′-O-methyl group, or additional 2′ chemicalmodification or a combination thereof.

In some embodiments, the mRNA comprises a poly A tail.

The compositions described herein, may be administered to a subject inneed thereof, in a therapeutically effective amount, to treat conditionsrelated to high circulating cholesterol levels. Conditions related tohigh circulating cholesterol level that may be treated using thecompositions and methods described herein include, without limitation:hypercholesterolemia, elevated total cholesterol levels, elevatedlow-density lipoprotein (LDL) levels, elevated blood LDL-cholesterollevels, reduced blood high-density lipoprotein cholesterol level, liversteatosis, coronary heart disease, vascular disease, ischemia, stroke,peripheral vascular disease, thrombosis, type 2 diabetes,hypertriglyceridemia, high elevated blood pressure, atherosclerosis,obesity, Alzheimer's disease, neurodegeneration, and combinationsthereof. The compositions and methods disclosed herein are effective inreducing the circulating cholesterol level in the subject, thus treatingthe conditions. The compositions and methods disclosed herein areeffective in reducing blood LDL cholesterol level and/or a reducingblood triglycerides level as compared to before the administration.

In another aspect, provided herein is a method for treating orpreventing a condition in a subject in need thereof, the methodcomprising administering to the subject the complex as provided herein,the composition as provided herein, or the lipid nanoparticle asprovided herein, wherein the sgRNA directs the adenosine base editorprotein to effect a modification in a target polynucleotide sequence ina cell of the subject, thereby treating or preventing the condition.

In some embodiments, the target polynucleotide sequence is in a PCSK9gene. In some embodiments, the modification reduces expression offunctional PCSK9 protein encoded by the PCSK9 gene in the subject. Insome embodiments, the condition is atherosclerotic vascular disease. Insome embodiments, the target polynucleotide sequence is in an ANGPTL3gene. In some embodiments, the modification reduces expression offunctional ANGPTL3 protein encoded by the ANGPTL3 gene in the subject.In some embodiments, the condition is an atherosclerotic vasculardisease, hypertriglyceridemia, or diabetes.

A patient who is being treated for a condition, a disease or a disorderis one who a medical practitioner has diagnosed as having such acondition. Diagnosis may be by any suitable means. Diagnosis andmonitoring may involve, for example, detecting the presence of diseased,dying or dead cells in a biological sample (e.g., tissue biopsy, bloodtest, or urine test), detecting the presence of plaques, detecting thelevel of a surrogate marker in a biological sample, or detectingsymptoms associated with a condition. A patient in whom the developmentof a condition is being prevented may or may not have received such adiagnosis. One in the art will understand that these patients may havebeen subjected to the same standard tests as described above or may havebeen identified, without examination, as one at high risk due to thepresence of one or more risk factors (e.g., family history or geneticpredisposition).

The therapeutic methods of the disclosure may be carried out on subjectsdisplaying pathology resulting from a disease or a condition, subjectssuspected of displaying pathology resulting from a disease or acondition, and subjects at risk of displaying pathology resulting from adisease or a condition. For example, subjects that have a geneticpredisposition to a disease or a condition can be treatedprophylactically. Subjects exhibiting symptoms associated with acondition, a disease or a disorder may be treated to decrease thesymptoms or to slow down or prevent further progression of the symptoms.The physical changes associated with the increasing severity of adisease or a condition are shown herein to be progressive. Thus, inembodiments of the disclosure, subjects exhibiting mild signs of thepathology associated with a condition or a disease may be treated toimprove the symptoms and/or prevent further progression of the symptoms.

In some embodiments, the subject exhibits a reduced blood LDLcholesterol level and/or a reduced blood triglycerides level as comparedto before the administration.

In some embodiments, after the administration, the subject exhibits areduced blood low-density lipoprotein (LDL) cholesterol level by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% ascompared to before the administration. In some embodiments, after theadministration, the subject exhibits a reduced blood low-densitylipoprotein (LDL) cholesterol level by at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, atleast 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, atleast 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, atleast 100 fold, at least 200 fold, at least 300 fold, at least 400 fold,at least 500 fold, at least 600 fold, at least 700 fold, at least 800fold, at least 900 fold, at least 1000 fold, at least 2000 fold, atleast 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or atleast 10000 fold as compared to before the administration.

In some embodiments, after the administration, the subject exhibits areduced blood triglycerides level by at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 98%, at least 99%, at least99.5%, at least 99.9%, or 100% as compared to before the administration.

In some embodiments, after the administration, the subject exhibits areduced blood triglycerides level by at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 97%, at least 98%, at least 99%, at least99.5%, at least 99.9%, or 100% as compared to before the administration.In some embodiments, after the administration, the subject exhibits areduced blood triglycerides level by at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, atleast 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, atleast 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, atleast 100 fold, at least 200 fold, at least 300 fold, at least 400 fold,at least 500 fold, at least 600 fold, at least 700 fold, at least 800fold, at least 900 fold, at least 1000 fold, at least 2000 fold, atleast 3000 fold, at least 4000 fold, at least 5000 fold, at least 6000fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, or atleast 10000 fold as compared to before the administration.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Adjustment andmanipulation of established dosages (e.g., frequency and duration) arewell within the ability of those skilled in the art. The treatment, suchas those disclosed herein, can be administered to the subject on adaily, twice daily, biweekly, monthly or any applicable basis that istherapeutically effective. In embodiments, the treatment is only on anas-needed basis, e.g., upon appearance of signs or symptoms of acondition or a disease, e.g., an atherosclerotic vascular disease,hypertriglyceridemia, or diabetes.

The compositions described herein, may be administered to a subject inneed thereof, in a therapeutically effective amount, to treat conditionsrelated to high circulating cholesterol levels. Conditions related tohigh circulating cholesterol level that may be treated using thecompositions and methods described herein include, without limitation:hypercholesterolemia, elevated total cholesterol levels, elevatedlow-density lipoprotein (LDL) levels, elevated blood LDL-cholesterollevels, reduced blood high-density lipoprotein cholesterol level, liversteatosis, coronary heart disease, vascular disease, ischemia, stroke,peripheral vascular disease, thrombosis, type 2 diabetes,hypertriglyceridemia, high elevated blood pressure, atherosclerosis,obesity, Alzheimer's disease, neurodegeneration, and combinationsthereof. The compositions and methods disclosed herein are effective inreducing the circulating cholesterol level in the subject, thus treatingthe conditions. The compositions and methods disclosed herein areeffective in reducing blood LDL cholesterol level and/or a reducingblood triglycerides level as compared to before the administration.

Toxicity and therapeutic efficacy of the compositions of the disclosurecan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects (the ratio LD50/ED50) is the therapeutic index.Agents that exhibit high therapeutic indices are preferred. The dosageof agents lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. While agents thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The skilled artisan will appreciate that certain factors may influencethe dosage and frequency of administration required to effectively treata subject, including but not limited to the severity of the disease ordisorder, previous treatments, the general characteristics of thesubject including health, sex, weight and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of the compositions can include asingle treatment or, preferably, can include a series of treatments. Itwill also be appreciated that the effective dosage of the composition ofthe disclosure used for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein. The therapeutically-effective dosage will generally be dependenton the patient's status at the time of administration. The preciseamount can be determined by routine experimentation but may ultimatelylie with the judgment of the clinician, for example, by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

Frequency of administration may be determined and adjusted over thecourse of therapy, and is generally, but not necessarily, based ontreatment and/or suppression and/or amelioration and/or delay of adisease. Alternatively, sustained continuous release formulations of apolypeptide or a polynucleotide may be appropriate. Various formulationsand devices for achieving sustained release are known in the art. Insome embodiments, dosage is daily, every other day, every three days,every four days, every five days, or every six days. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays.

The dosing regimen (including a composition disclosed herein) can varyover time. In some embodiments, for an adult subject of normal weight,doses ranging from about 0.01 to 1000 mg/kg may be administered. In someembodiments, the dose is between 1 to 200 mg. The particular dosageregimen, i.e., dose, timing and repetition, will depend on theparticular subject and that subject's medical history, as well as theproperties of the polypeptide or the polynucleotide (such as thehalf-life of the polypeptide or the polynucleotide, and otherconsiderations well known in the art).

For the purpose of the present disclosure, the appropriate therapeuticdosage of a composition as described herein will depend on the specificagent (or compositions thereof) employed, the formulation and route ofadministration, the type and severity of the disease, whether thepolypeptide or the polynucleotide is administered for preventive ortherapeutic purposes, previous therapy, the subject's clinical historyand response to the antagonist, and the discretion of the attendingphysician. Typically, the clinician will administer a polypeptide untila dosage is reached that achieves the desired result.

Administration of one or more compositions can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of a composition may be essentially continuous over apreselected period of time or may be in a series of spaced dose, e.g.,either before, during, or after developing a disease.

The methods and compositions of the disclosure described hereinincluding embodiments thereof can be administered with one or moreadditional therapeutic regimens or agents or treatments, which can beco-administered to the mammal.

Metabolic Syndrome and Cardiovascular Disease

The ability of gene modification, especially to edit bases directlyallows for the precise edit, modification, and/or disruption of genes invivo without the need to create DSBs and improved methods of treatinghuman disease. For example, Chadwick et. al used cytosine base editingto introduce nonsense mutations into the Pcsk9 gene in adult mice.Delivery of BE3 and a gRNA via adenoviral vector into the liver caused areduction in plasma PCSK9 levels by 56%. Furthermore, cholesterol levelsin the base-edited mice were reduced by roughly 30%. In subsequent work,cytosine base editing to disrupt the Angptl3 gene in adult mice resultedin substantially reduced blood cholesterol and triglyceride levels, andcytosine base editing to disrupt the Pcsk9 gene or the Hpd gene in fetalmice resulted in postnatal reduction of cholesterol levels or cure ofthe disease hereditary tyrosinemia type 1.

Cardiovascular disease, obesity, type 2 diabetes, metabolic syndrome mayshare one or more similar underlying etiologies. The human PCSK9 geneencodes a protein that helps regulate the amount of cholesterol in thebloodstream. The liver protein Proprotein Convertase Subtilisin/KexinType 9 (PCSK9) in human is a secreted, globular, auto-activating serineprotease that acts as a protein-binding adaptor within endosomalvesicles to bridge a pH-dependent interaction with the low-densitylipoprotein receptor (LDL-R) during endocytosis of LDL particles,preventing recycling of the LDL-R to the cell surface and leading toreduction of LDL-cholesterol clearance. PCSK9 orthologs are found acrossmany species. The PCSK9 protein breaks down low-density lipoproteinreceptors before they reach the cell surface, so more cholesterol canremain in the bloodstream. As a result, the PCSK9 gene and the encodedPCSK9 protein are attractive targets for regulating cholesterolmetabolism, especially in lowering blood cholesterol levels.

Other targets have also been shown that are associated with decreasedplasma levels of triglycerides (TGs), low-density lipoproteincholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C),which leads to a significant reduction in cardiovascular risk. Forexample, human ANGPTL3 is considered an important new pharmacologicaltarget for the treatment of cardiovascular diseases. Experimentalevidence demonstrates that anti-ANGPTL3 therapies have an importantanti-atherosclerotic effect. Results from phase I clinical trials with amonoclonal anti-ANGPTL3 antibody (evinacumab) and anti-senseoligonucleotide (ASO) clearly show a significant lipid lowering effect.

Human ANGPTL3 gene is located on chromosome 1p31. Genetic variants ofhuman ANGPTL3 gene have been linked to different plasma lipid profiles.For example, homozygous loss of function (LOF) variants of ANGPTL3 genecause the levels of all plasma lipoproteins to be greatly reduced.ANGPTL3 is exclusively expressed in the liver and is secreted intocirculation where it goes through cleavage by hepatic proproteinconvertases. The ANGPTL3 protein is a 460-amino-acid (aa) polypeptidewith a distinctive signal peptide sequence, a N-terminal helical domainand a C-terminal globular fibrinogen homology domain. The N-terminalcoiled-coil region (17-207aa) affects plasma TG levels via reversiblyinhibiting catalytic activity of LPL while the fibrinogen-like domain(207-460aa) binds to integrin αvβ3 receptor and affects angiogenesis,which is similar to the function of angiopoietins. A short linker region(at 221-222 and 224-225) between N- and C-terminal domains is a specialzone, which has been verified to be split by furin. Existed results haveshown that the truncated form of cleavage ANGPTL3 could reinforce theinhibitory activity of LPL and endothelial lipase (EL), suggesting thatthe cleavage type of ANGPTL3 may function more effectively.

Moreover, human apolipoprotein C-III (APOC3) could potentially beanother therapeutic target. APOC3 is a protein that in humans is encodedby the APOC3 gene. APOC3 is a component of VLDL. APOC3 inhibitslipoprotein lipase and hepatic lipase. It is also thought to inhibithepatic uptake of triglyceride-rich particles. An increase in APOC3levels induces the development of hypertriglyceridemia. Recent evidencesuggests an intracellular role for APOC3 in promoting the assembly andsecretion of triglyceride-rich VLDL particles from hepatic cells underlipid-rich conditions. However, two naturally occurring point mutationsin human apoC3 coding sequence, A23T and K58E have been shown to abolishthe intracellular assembly and secretion of triglyceride-rich VLDLparticles from hepatic cells.

Targeted base editing with target genes involved in vascular disease anddiabetes as described in Chadwick A C, Wang X, Musunuru K. In vivo baseediting of PCSK9 (proprotein convertase subtilisin/kexin type 9) as atherapeutic alternative to genome editing. Arterioscler Thromb VascBiol, 2017, 37: 1741-7; Chadwick A C, Evitt N H, Lv W, et al. Reducedblood lipid levels with in vivo CRISPR-Cas9 base editing of ANGPTL3.Circulation, 2018, 137: 975-7; Rossidis A C, Stratigis J D, Chadwick AC, et al. In utero CRISPR-mediated therapeutic editing of metabolicgenes. Nat Med, 2018, 24: 1513-8 is incorporated herein by reference inits entirety. While genes involved in lipid metabolism, including PCSK9,have been edited by targeted by current gene editing technologies, thereremains a need for precise base editing targeting these genes forimproved editing outcomes.

Pharmaceutical Composition

In some aspects, provided herein is a pharmaceutical compositioncomprising the base editor system as provided herein and apharmaceutically acceptable carrier or excipient.

In some aspects, provided herein, is a pharmaceutical composition forgene modification comprising a gRNA or sgRNA described herein and a Baseeditor fusion protein or a nucleic acid sequence encoding the Baseeditor fusion protein and a pharmaceutically acceptable carrier. Thecomposition for gene modification comprising a gRNA or sgRNA describedherein and a Base editor fusion protein or a nucleic acid sequenceencoding the Base editor fusion protein can be formulated intopharmaceutical compositions. Pharmaceutical compositions are formulatedin a conventional manner using one or more pharmaceutically acceptableinactive ingredients that facilitate processing of the active compoundsinto preparations that can be used pharmaceutically. Suitableformulations for use in the present disclosure and methods of deliveryare generally well known in the art. Proper formulation is dependentupon the route of administration chosen. A summary of pharmaceuticalcompositions described herein can be found, for example, in Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated byreference for such disclosure.

A pharmaceutical composition can be a mixture of a gRNA or sgRNAdescribed herein and a Base editor fusion protein or a nucleic acidsequence encoding the Base editor fusion protein with one or more ofother chemical components (i.e., pharmaceutically acceptableingredients), such as carriers, excipients, binders, filling agents,suspending agents, flavoring agents, sweetening agents, disintegratingagents, dispersing agents, surfactants, lubricants, colorants, diluents,solubilizers, moistening agents, plasticizers, stabilizers, penetrationenhancers, wetting agents, anti-foaming agents, antioxidants,preservatives, or one or more combination thereof. The pharmaceuticalcomposition facilitates administration of the gRNA or sgRNA describedherein and the Base editor fusion protein or a nucleic acid sequenceencoding the Base editor fusion protein to an organism or a subject inneed thereof.

The pharmaceutical compositions of the present disclosure can beadministered to a subject using any suitable methods known in the art.The pharmaceutical compositions described herein can be administered tothe subject in a variety of ways, including parenterally, intravenously,intradermally, intramuscularly, colonically, rectally, orintraperitoneally. In some embodiments, the pharmaceutical compositionscan be administered by intraperitoneal injection, intramuscularinjection, subcutaneous injection, or intravenous injection of thesubject. In some embodiments, the pharmaceutical compositions can beadministered parenterally, intravenously, intramuscularly, or orally.

For administration by inhalation, the adenovirus described herein can beformulated for use as an aerosol, a mist, or a powder. For buccal orsublingual administration, the pharmaceutical compositions may beformulated in the form of tablets, lozenges, or gels formulated in aconventional manner. In some embodiments, the adenovirus describedherein can be prepared as transdermal dosage forms. In some embodiments,the adenovirus described herein can be formulated into a pharmaceuticalcomposition suitable for intramuscular, subcutaneous, or intravenousinjection. In some embodiments, the adenovirus described herein can beadministered topically and can be formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams, or ointments. In someembodiments, the adenovirus described herein can be formulated in rectalcompositions such as enemas, rectal gels, rectal foams, rectal aerosols,suppositories, jelly suppositories, or retention enemas. In someembodiments, the adenovirus described herein can be formulated for oraladministration such as a tablet, a capsule, or liquid in the form ofaqueous suspensions or solutions selected from the group including, butnot limited to, aqueous oral dispersions, emulsions, solutions, elixirs,gels, and syrups.

In some embodiments, the pharmaceutical composition for genemodification comprising a gRNA or sgRNA described herein and a Type IICas protein or a nucleic acid sequence encoding the Type II Cas proteinfurther comprises a therapeutic agent. The additional therapeutic agentmay modulate different aspects of the disease, disorder, or conditionbeing treated and provide a greater overall benefit than administrationof either the replication competent recombinant adenovirus or thetherapeutic agent alone. Therapeutic agents include, but are not limitedto, a chemotherapeutic agent, a radiotherapeutic agent, a hormonaltherapeutic agent, and/or an immunotherapeutic agent. In someembodiments, the therapeutic agent may be a radiotherapeutic agent. Insome embodiments, the therapeutic agent may be a hormonal therapeuticagent. In some embodiments, the therapeutic agent may be animmunotherapeutic agent. In some embodiments, the therapeutic agent is achemotherapeutic agent. Preparation and dosing schedules for additionaltherapeutic agents can be used according to manufacturers' instructionsor as determined empirically by a skilled practitioner. For example,preparation and dosing schedules for chemotherapy are also described inThe Chemotherapy Source Book, 4th Edition, 2008, M. C. Perry, Editor,Lippincott, Williams & Wilkins, Philadelphia, Pa.

The subjects that can be treated with gene modification compositions ofchemically modified gRNAs or sgRNAs described herein and a Type II Casprotein or a nucleic acid sequence encoding the Type II Cas protein andmethods described herein can be any subject with a disease or acondition. For example, the subject may be a eukaryotic subject, such asan animal. In some embodiments, the subject is a mammal, e.g., human. Insome embodiments, the subject is a human. In some embodiments, thesubject is a non-human animal. In some embodiments, the subject is afetus, an embryo, or a child. In some embodiments, the subject is anon-human primate such as chimpanzee, and other apes and monkey species;farm animals such as cattle, horses, sheep, goats, pigs; domesticanimals such as rabbits, dogs, and cats; laboratory animals includingrodents, such as rats, mice, and guinea pigs, and the like.

In some embodiments, the subject is prenatal (e.g., a fetus), a child(e.g., a neonate, an infant, a toddler, a preadolescent), an adolescent,a pubescent, or an adult (e.g., an early adult, a middle-aged adult, asenior citizen). The human subject can be between about 0 month andabout 120 years old, or older. The human subject can be between about 0and about 12 months old; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 months old. The human subject can be between about 0 and12 years old; for example, between about 0 and 30 days old; betweenabout 1 month and 12 months old; between about 1 year and 3 years old;between about 4 years and 5 years old; between about 4 years and 12years old; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old. Thehuman subject can be between about 13 years and 19 years old; forexample, about 13, 14, 15, 16, 17, 18, or 19 years old. The humansubject can be between about 20 and about 39 years old; for example,about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, or 39 years old. The human subject can be between about 40to about 59 years old; for example, about 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old. Thehuman subject can be greater than 59 years old; for example, about 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, or 120 years old. The humansubjects can include male subjects and/or female subjects.

Lipid Nanoparticle (LNP) Composition

In some embodiments, LNPs are prepared in accordance with the methodsdescribed in Conway, A. et al. 2019 Mol. Ther. 27, 866-877, andVilliger, L. et al 2021 Nat. Biomed. Eng. 5, 179-18, which areincorporated by reference. In some embodiments, LNPs are composed of anamino lipid, a monomethoxypolyethylene glycol (or methoxypoluyethyleglycol) of average molecular weight 2000 Da conjugated to a lipid calledPEG-Lipid, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC). In some embodiments, LNPs are composed of proprietary ionizablecationic lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol,and a PEG-lipid.

In some embodiments, LNPs have an average hydrodynamic diameter of about30 about 160, about 35-about 160, about 40-about 160, about 45-about160, about 50-about 160, about 55-about 160, about 60-about 160, about65-about 160, about 70-about 160, about 75-about 160, about 80-about160, about 85-about 160, about 90-about 160, about 95-about 160, about100-about 160, about 105-about 160, about 110-about 160, about 115-about160, about 120-about 160, about 125-about 160, about 130-about 160,about 135-about 160, about 140-about 160, about 145-about 160, about150-about 160, about 30-about 155, about 30-about 150, about 30-about145, about 30-about 140, about 30 about 135, about 30-about 130, about30-about 125, about 30-about 120, about 30-about 115, about 30-about110, about 30-about 105, about 30-about 100, about 30-about 95, about30-about 90, about 30-about 85, about 30-about 80, about 30-about 75,about 30 about 70, about 30-about 65, about 30-about 60, about 30-about55, about 30-about 50, about 30-about 45, about 30-about 40, about30-about 45, about 35-about 45, about 35 about 50, about 40-about 50,about 40-about 55, about 45-about 55, about 45-about 60, about 50-about60, about 50-about 65, about 55-about 60, about 55-about 65, about 55about 70, about 60-about 70, about 60-about 75, about 65-about 75, about65-about 80, about 70-about 80, about 70-about 85, about 75-about 85,about 75-about 90, about 80 about 90, about 80-about 95, about 85-about95, about 85-about 100, about 90-about 100, about 90-about 105, about95-about 105, about 95-about 110, about 100-about 110, about 100-about115, about 105-about 115, about 105-about 120, about 110-about 120,about 110-about 125, about 115-about 125, about 115-about 130, about120-about 130, about 120-about 135, about 125-about 135, about 125-about140, about 130-about 140, about 130-about 145, about 35-about 140, about45-about 130, about 55-about 120, about 65-about 110, about 75-about100, or about 85-about 90 nm.

In some embodiments, LNPs comprise about 1-about 97, about 5-about 97,about 10-about 97, about 15-about 97, about 20-about 97, about 25-about97, about 30-about 97, about 35-about 97, about 40-about 97, about45-about 97, about 50-about 97, about 55-about 97, about 60-about 97,about 65-about 97, about 70-about 97, about 75- about 97, about 80-about97, about 1-about 95, about 1-about 90, about 1-about 85, about 1 about80, about 1-about 75, about 1-about 70, about 1-about 65, about 1-about60, about 1-about 55, about 1-about 50, about 1-about 45, about 1-about40, about 1-about 35, about 1-about 30, about 1-about 25, about 1-about20, about 1-about 15, about 1-about 10, about 10-about 30, about10-about 35, about 15-about 35, about 15-about 40, about 20-about 40,about 20-about 45, about 25-about 45, about 25-about 50, about 30-about50, about 30-about 55, about 35-about 55, about 35-about 60, about40-about 60, about 40-about 65, about 45-about 65, about 45-about 70,about 50-about 70, about 50-about 75, about 55-about 75, about 55-about80, or about 60-about 80% of amino lipids (in mol %).

In some embodiments, LNPs comprise about 1-about 40, about 1-about 38,about 1-about 36, about 1-about 34, about 1-about 32, about 1-about 30,about 1-about 28, about 1-about 26, about 1-about 24, about 1-about 22,about 1-about 20, about 1-about 18, about 1-about 16, about 1-about 14,about 1-about 12, about 1-about 10, about 1 about 8, about 1-about 6,about 1-about 4, about 1-about 2, about 2-about 40, about 4 about 40,about 6-about 40, about 8-about 40, about 10-about 40, about 12-about40, about 14-about 40, about 16-about 40, about 18-about 40, about20-about 40, about 22 about 40, about 24-about 40, about 26-about 40,about 28-about 40, about 30-about 40, about 32-about 40, about 34-about40, about 36-about 40, about 38-about 40, about 2 about 35, about2-about 30, about 2-about 25, about 2-about 20, about 2-about 15, about2-about 10, about 2-about 8, about 2-about 6, or about 2-about 4% DSPC(in mol %).

In some embodiments, LNPs comprise about 1-about 20, about 1-about 19,about 1-about 18, about 1-about 17, about 1-about 16, about 1-about 15,about 1-about 14, about 1-about 13, about 1-about 12, about 1-about 11,about 1-about 10, about 1-about 9, about 1-about 8, about 1-about 7,about 1-about 6, about 1-about 5, about 1-about 4, about 1-about 3,about 2-about 6, about 3-about 7, about 4-about 8, about 5-about 9,about 6-about 10, about 7-about 11, about 8-about 12, about 9-about 13,about 10 about 14, about 11-about 15, about 12-about 16, about 13-about17, about 14-about 18, about 15-about 19, or about 16-about 20%PEG-Lipid (in mol %),

In some embodiments, LNPs comprise about 1-about 97, about 5-about 97,about 10-about 97, about 15-about 97, about 20-about 97, about 25-about97, about 30-about 97, about 35-about 97, about 40-about 97, about45-about 97, about 50-about 97, about 55-about 97, about 60-about 97,about 65-about 97, about 70-about 97, about 75-about 97, about 80-about97, about 1-about 95, about 1-about 90, about 1-about 85, about 1 about80, about 1-about 75, about 1-about 70, about 1-about 65, about 1-about60, about 1-about 55, about 1-about 50, about 1-about 45, about 1-about40, about 1-about 35, about 1-about 30, about 1-about 25, about 1-about20, about 1-about 15, about 1-about 10, about 10-about 30, about10-about 35, about 15-about 35, about 15-about 40, about 20-about 40,about 20-about 45, about 25-about 45, about 25-about 50, about 30-about50, about 30-about 55, about 35-about 55, about 35-about 60, about40-about 60, about 40-about 65, about 45-about 65, about 45-about 70,about 50-about 70, about 50-about 75, about 55-about 75, about 55-about80, or about 60-about 80% of cholesterol (in mol %).

In some embodiments, LNPs comprise about 1-about 97, 5-about 97,10-about 97, 15-about 97, 20-about 97, 25-about 97, 30-about 97,35-about 97, 40-about 97, 45 about 97, 50-about 97, 55-about 97,60-about 97, 65-about 97, 70-about 97, 75-about 97, 80-about 97, 1-about95, 1-about 90, 1-about 85, 1-about 80, 1-about 75, 1-about 70, 1-about65, 1-about 60, 1-about 55, 1-about 50, 1-about 45, 1-about 40, 1-about35, 1-about 30, 1-about 25, 1-about 20, 1-about 15, 1-about 10, 10-about30, 10 about 35, 15-about 35, 15-about 40, 20-about 40, 20-about 45,25-about 45, 25-about 50, 30-about 50, 30-about 55, 35-about 55,35-about 60, 40-about 60, 40-about 65, 45-about 65, 45-about 70,50-about 70, 50-about 75, 55-about 75, 55-about 80, or 60 about 80% ofamino lipids; 1-about 40, 1-about 38, 1-about 36, 1-about 34, 1-about32, 1-about 30, 1-about 28, 1-about 26, 1-about 24, 1-about 22, 1-about20, 1-about 18, 1-about 16, 1-about 14, 1-about 12, 1-about 10, 1-about8, 1-about 6, 1-about 4, 1 about 2, 2-about 40, 4-about 40, 6-about 40,8-about 40, 10-about 40, 12-about 40, 14-about 40, 16-about 40, 18-about40, 20-about 40, 22-about 40, 24-about 40, 26 about 40, 28-about 40,30-about 40, 32-about 40, 34-about 40, 36-about 40, 38-about 40, 2-about35, 2-about 30, 2-about 25, 2-about 20, 2-about 15, 2-about 10, 2-about8, 2-about 6, or 2-about 4% DSPC; 1-about 20, 1-about 19, 1-about 18,1-about 17, 1 about 16, 1-about 15, 1-about 14, 1-about 13, 1-about 12,1-about 11, 1-about 10, 1 about 9, 1-about 8, 1-about 7, 1-about 6,1-about 5, 1-about 4, 1-about 3, 2-about 6, 3-about 7, 4-about 8,5-about 9, 6-about 10, 7-about 11, 8-about 12, 9-about 13, 10 about 14,11-about 15, 12-about 16, 13-about 17, 14-about 18, 15-about 19, andabout 16-about 20% PEG-Lipid, with the balance being cholesterol (all inmol %).

Amino Lipid

Described herein are LNP compositions comprising an amino lipid, aphospholipid, a PEGlipid, a cholesterol or a derivative thereof, apayload, or any combination thereof. In some embodiments, the LNPcomposition comprises an amino lipid. In one aspect, disclosed herein isan amino lipid having the structure of Formula (I), or apharmaceutically acceptable salt or solvate thereof,

whereineach of R¹ and R² is independently C₇-C₂₂ alkyl, C₇-C₂₂ alkenyl, C₃-C₈cycloalkyl, —C₂-C₁₀ allkylene-L-R⁶, or

wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl isindependently substituted or unsubstituted;each of X, Y, and Z is independently —C(═O)NR⁴—, —NR⁴C(═O)—, —C(═O)O—,—OC(═O)—, —OC(═O)O—, —NR⁴C(═O)O—, —OC(═O)NR⁴—, —NR⁴C(═O)NR⁴—,—NR⁴C(═NR⁴)NR⁴—, —C(═S)NR⁴—, —NR⁴C(═S)—, —C(═O)O—, —OC(═S)—, OC(═S)O—,—NR⁴C(═S)O—, —OC(═S)NR⁴—, —NR⁴C(═S)NR⁴—, —C(═O)S—, —SC(═O)—, —OC(═O)S—,—NR⁴C(═O)S—. —SC(═O)NR⁴—, —C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR⁴C(═S)S—,—SC(═S)NR⁴—, —C(═S)S—, —SC(═S)—, —SC(═O)S—, —SC(═S)S—, —NR⁴C(═S)S—,—SC(═S)NR⁴—O, S, or a bond;each of L is independently —C(═O)NR⁴—, —NR⁴C(═O)—, —C(═O)O—. —OC(═O)O—,—NR⁴C(═O)O—, —OC(═O)NR⁴—, —NR⁴C(═O)NR⁴—, —NR⁴C(═NR⁴)NR⁴—, —C(═S)NR⁴—,—NR⁴C(═S)—, —C(═O)O—, —OC(═S)—, OC(═S)O—, —NR⁴C(═S)O—, —OC(═S)NR⁴—,—NR⁴C(═S)NR⁴—, —C(═O)S—, SC(═O)—, —OC(═O)S—, —NR⁴C(═O)S—, —SC(═O)NR⁴—,—C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR⁴C(═S)S—, —SC(═S)NR⁴—, —C(═S)S—,—SC(═S)—, —SC(═O)S—, —SC(═S)S—, —NR⁴C(═S)S—, —SC(═S)NR⁴—, O, S, —C₁-C₁₀alkylene-O—, —C₁-C₁₀ alkylene-C(═O)O—, —C₁-C₁₀ alkylene-OC(═O)—, or abond, wherein the alkylene is substituted or unsubstituted;R³ is —C₀-C₁₀ alkylene-NR⁷R⁸, —C₀-C₁₀ alkylene-heterocycloalkyl, or—C₀-C₁₀ alkylene-heterocycloaryl,wherein the alkylene, heterocycloalkyl and heterocycloaiyl isindependently substituted or unsubstituted; each of R⁴ is independentlyhydrogen or substituted or unsubstituted C₁-C₆ alkyl;R⁵ is hydrogen or substituted or unsubstituted C₁-C₆ alkyl;each of R⁶ is independently substituted or unsubstituted C₃-C₂₂ alkyl orsubstituted or unsubstituted C₃-C₂₂ alkenyl;each of R⁷ and R⁸ is independently hydrogen or substituted orunsubstituted C₁-C₆ alkyl, orR⁷ and R⁸ taken together with the nitrogen to which they are attachedform a substituted or unsubstituted C₂-C₆ heterocyclyl;p is an integer selected from 1 to 10; andeach of n, m, and q is independently 0, 1, 2, 3, 4, or 5.

In some embodiments of Formula (I), if the structure carries more thanone asymmetric C-atom, each asymmetric C-atom independently representsracemic, chirally pure R and/or chirally pure S isomer, or a combinationthereof.

In some embodiments, each of n, in, and q in Formula (I) isindependently 0, 1, 2, or 3. In some embodiments, each of n, m, and q inFormula (I) is 1.

In some embodiments, the compound of Formula (1) has a structure ofFormula (Ia), or a pharmaceutically acceptable salt or pharmaceuticallyacceptable solvate thereof:

whereineach of R¹ and R² is independently C₇-C₂₂ alkyl, C₇-C₂₂ alkenyl, C₃-C₈cycloalkyl, —C₂-C₁₀ alkylene-L-R⁶, or

wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl isindependently substituted or unsubstituted;each of X, Y, and Z is independently C(═O)NR⁴—, —NR⁴C(D)—, —C(═O)O—,—OC(═O)—, —OC(═O)O—, —NR⁴C(═O)O—, —OC(═O)NR⁴—, —NR⁴C═O)NR⁴—,—NR⁴C(═NR⁴)NR⁴—. —C(═S)NR⁴—, —NR⁴C(═S)—, —C(E)O—, —OC(═S)—, OC(═S)O—,—NR⁴C(═S)O—, —OC(═S)NR⁴—, —NR⁴C(═S)NR⁴—, —C(═O)S—, —SC(═O)—, —OC(═O)S—,—NR⁴C(═O)S—, —SC(═O)NR⁴— —C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR⁴C(═S)S—,—SC(═S)NR⁴—, —C(═S)S—. —SC(═S)—, —SC(═O)S—, —SC(═S)S—. —NR⁴C(═S)S—,—SC(═S)NR⁴—, O, S, —C₁-C₁₀ alkylene-O—, or a bond, wherein the alkyleneis substituted or unsubstituted;each of L is independently —C(═O)NR⁴—, —NR⁴C(═O)—, —C(═O)O—, —OC(═O)—,—OC(═O)O—, —NR⁴C(═O)O—, —OC(═O)NR⁴—, —NR⁴C(═O)NR⁴—, —NR⁴C(═NR⁴)NR⁴—.—C(═S)NR⁴—, —NR⁴C(═S)—, —C(═O)O—, —OC(═S)—, OC(═S)O—, —NR⁴C(═S)O—,—OC(═S)NR⁴—, —NR⁴C(═S)NR⁴—, —C(═O)S—, —SC(═O)—, —OC(═O)S—, —NR⁴C(═O)S—,—SC(═O)NR⁴— —C(═S)S—, —SC(═S)—, —SC(═S)O—, —NR⁴C(═S)S—, —SC(═S)NR⁴—,—C(═S)S—, —SC(═S)—, —SC(═O)S—, —SC(═S)S—, —NR⁴C(═S)S—, —SC(═S)NR⁴—, O,S. —C₁-C₁₀ alkylene-O—, —C₁-C₁₀ alkylene-C(═O)O—, —C₁-C₁₀ alkylene-OC(═O)—, or a bond, wherein the alkylene is substituted orunsubstituted;R³ is —C₀-C₁₀ alkylene-NR⁷R⁸, —C₀-C₁₀ alkylene-heterocycloalkyl, or—C₀-C₁₀ alkylene-heterocyclowyl, wherein the alkylene, heterocycloalkyland heterocycloaryl is independently substituted or unsubstituted;each of R⁴ is independently hydrogen or substituted or unsubstitutedCI—C6 alkyl;R⁵ is hydrogen or substituted or unsubstituted C₁-C₆ alkyl;each of R⁶ is independently substituted or unsubstituted C₃-C₂₂ alkyl orsubstituted or unsubstituted C₃-C₂₂ alkenyl;each of R⁷ and R⁸ is independently hydrogen or substituted orunsubstituted C₁-C₆ alkyl, orR⁷ and R⁸ taken together with the nitrogen to which they are attachedform a substituted or unsubstituted C₂-C₆ heterocyclyl; andp is an integer selected from 1 to 10.

In some embodiments of Formula (1a), if the structure carries more thanone asymmetric Catom, each asymmetric C-atom independently representsracemic, chirally pure R and/or chirally pure S isomer, or a combinationthereof.

In some embodiments, R¹ and R² in Formula (I) and Formula (Ia) isindependently C₇-C₂₂ alkyl, C₇-C₂₂ alkenyl, —C₂-C₁₀ alkylene-L-R⁶, or

wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl isindependently substituted or unsubstituted. In some embodiments, R¹ andR² in Formula (I) and Formula (1a) is independently C₁₀-C₂₀ alkyl,C₁₀-C₂₀ alkenyl. —C₈-C₇ alkylene-L-R⁶, or

wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl isindependently substituted or unsubstituted. In some embodiments, R¹ inFormula (I) and Formula (1a) is

In some embodiments, each of L in Formula (I) and Formula (Ia) isindependently O, S, —C₁-C₁₀ alkylene-O—, —C₁-C₁₀ alkylene-C(═O)O—,—C₁-C₁₀ alkylene-OC(═O)—, or a bond, wherein the alkylene is substitutedor unsubstituted. In some embodiments, each of L in Formula (I) andFormula (Ia) is independently O, S, —C₁-C₃ alkylene-O—, —C₁-C₃alkylene-C(═O)O—, —C₁-C₃ alkylene-OC(═O)—, or a bond, wherein thealkylene is substituted or unsubstituted. In some embodiments, each of Lin Formula (I) and Formula (1a) is independently O, S, —C₁-C₃alkylene-O—, —C₁-C₃ alkylene-C(═O)O—, —C₁-C₃ alkylene-OC(═O)—, or abond, wherein the alkylene is linear or branched unsubstituted alkylene.

In some embodiments, each of R⁶ in Formula (I) and Formula (1a) isindependently substituted or unsubstituted linear C₃-C₂₂ alkyl orsubstituted or unsubstituted linear C₃-C₂₂ alkenyl. In some embodiments,each of R⁶ in Formula (I) and Formula (Ia) is independently substitutedor unsubstituted C₃-C₂₀ alkyl or substituted or unsubstituted C₃-C₂₀alkenyl. In some embodiments, each of R⁶ in Formula (I) and Formula (Ia)is independently substituted or unsubstituted C₃-C₁₀ alkyl orsubstituted or unsubstituted C₃-C₁₀ alkenyl. In some embodiments, eachof R⁶ in Formula (I) and Formula (Ia) is independently substituted orunsubstituted C₃-C₁₀ alkyl. In some embodiments, each of R⁶ in Formula(I) and Formula (1a) is independently substituted or unsubstitutedlinear C₃-C₁₀ alkyl. In some embodiments, each of R⁶ in Formula (I) andFormula (Ia) is independently substituted or unsubstituted n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, or n-dodecyl.In some embodiments, each of R⁶ in Formula (I) and Formula (Ia) isindependently substituted or unsubstituted n-octyl. In some embodiments,each of R⁶ in Formula (I) and Formula (Ia) is n-octyl.

In some embodiments, each of L in Formula (I) and Formula (Ia) isindependently —C(═O)O—, —OC(═O)—, —C₁-C₁₀ alkylene-O—, or O. In someembodiments, each of L in Formula (I) and Formula (Ia) is O. In someembodiments, each of L in Formula (I) and Formula (Ia) is —C₁-C₃alkylene-O—. In some embodiments, p in Formula (I) and Formula (1a) is1, 2, 3, 4, or 5. In some embodiments, p in Formula (I) and Formula (Ia)is 2.

In some embodiments, R¹ in Formula (I) and Formula (Ia) is

In some embodiments, each of R⁴ in Formula (I) and Formula (Ia) isindependently H or substituted or unsubstituted C₁-C₄ alkyl. In someembodiments, each of. R⁴ in Formula (I) and Formula (1a) isindependently substituted or unsubstituted linear C₁-C₄ alkyl. In someembodiments, each of R⁴ in Formula (1) and Formula (1a) is H. In someembodiments, each of R⁴ in Formula (I) and Formula (Ia) is independentlyH, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, or —CH(CH₃)₂. In some embodiments, each ofR⁴ in Formula (I) and Formula (Ia) is independently H or —CH₃. In someembodiments, each of R⁴ in Formula (1) and Formula (Ia) is —CH₃.

In some embodiments, X in Formula (I) and Formula (Ia) is —C(═O)O— or—OC(═O))—. In some embodiments, X in Formula (I) and Formula (Ia) is—C(═O)NR⁴— or —NR⁴C(═O)—. In some embodiments, X in Formula (I) andFormula (Ia) is —C(═O)N(CH₃)—, —N(CH₃)C(═O)—, —C(═O)NH—, or —NHC(═O)—.In some embodiments, X in Formula (I) and Formula (Ia) is —C(═O))NH—,—C(═O)N(CH₃)—. —OC(═O))—, —NHC(═O)—, —N(CH₃)C(═O))—, —C(═O)O—,—OC(═O)O—, —NHC(═O)O—, —N(CH₃)C(═O)O—, —OC(═O))NH—, —OC(═O)N(CH₃)—,—NHC(═O)NH—, —N(CH₃)C(═O))NH—, —NHC(═O)N(CH₃)—, —N(CH₃)C(═O)N(CH₃)—,NHC(═NH)NH—, —N(CH₃)C(═NH)NH—, —NHC(═NH)N(CH₃)—, —N(CH₃)C(═NH)N(CH₃)—,NHC(═NMe)NH—, —N(CH₃)C(═NMe)NH—, —NHC(═NMe)N(CH₃)—, or—N(CH₃)C(═NMe)N(CH₃)—.

In some embodiments. R² in Formula (I) and Formula (Ia) is C₇-C₂₂ alkyl,C₇-C₂₂ alkenyl, —C₂-C₁₀ alkylene-L-R⁶, or

Wherein each of the alkyl, alkylene, alkenyl, and cycloalkyl isindependently substituted or unsubstituted. In some embodiments, R² inFormula (I) and Formula (Ia) is substituted or unsubstituted C₇-C₂₂alkyl or substituted or unsubstituted C₇-C₂₂ alkenyl. In someembodiments, R² in Formula (I) and Formula (1a) is substituted orunsubstituted linear C₇-C₂₂ alkyl or substituted or unsubstituted linearC₇-C₂₂ alkenyl. In some embodiments, R² in Formula (I) and Formula (1a)is substituted or unsubstituted C₁₀-C₂₀ alkyl or substituted orunsubstituted C₁₀-C₂₀ alkenyl. In some embodiments, R² in Formula (I)and Formula (Ia) is unsubstituted C₁₀-C₂₀ alkyl. In some embodiments, R²in Formula (I) and Formula (Ia) is unsubstituted C₁₀-C₂₀ alkenyl. Insome embodiments, R² in Formula (I) and Formula (1a) is —C₂-C₁₀alkylene-L-R⁶. In some embodiments, R² in Formula (I) and Formula (Ia)is —C₂-C₁₀ alkylene- C(═O)O— R⁶ or —C₂-C₁₀ alkylene-OC(═O)— R⁶.

In some embodiments, R² in Formula (I) and Formula (Ia) is

In some embodiments, Y in Formula (I) and Formula (Ia) is —C(═O)O— or—OC(═O)—. In some embodiments, Y in Formula (I) and Formula (Ia) is—C(═O)NR⁴— or —NR⁴C(═O)—. In some embodiments, Y in Formula (I) andFormula (Ia) is —C(═O)N(CH₃)—, —N(CH₃)C(═O)—, —C(═O)NH—, or —NHC(═O)—.In some embodiments, Y in Formula (I) and Formula (Ia) is —OC(═O)O—,—NR⁴C(═O)O—, —OC(═O)NR⁴—, or —NR⁴C(═O)NR⁴—. In some embodiments. Y inFormula (I) and Formula (Ia) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—,—NHC(═O)NH—, —N(CH₃)C(═O)O—. —OC(═O)N(CH₃)—, —N(CH₃)C(═O)N(CH₃)— or—N(CH₃)C(═O)NH—. In some embodiments, Y in Formula (I) and Formula (Ia)is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or —NHC(═O)NH—.

In some embodiments, R³ in Formula (I) and Formula (Ia) is —C₀-C₁₀alkylene-NR⁷R⁸ or —C₀-C₁₀ alkylene-heterocycloalkyl, wherein thealkylene and heterocycloalkyl is independently substituted orunsubstituted. In some embodiments, R³ in Formula (I) and Formula (Ta)is —C₀-C₁₀ alkylene-NR⁷R⁸. Tn some embodiments, R³ in Formula (I) andFormula (Ia) is —C₁-C₆ alkylene-NR⁷R⁸. In some embodiments, R³ inFormula (I) and Formula (Ia) is —C₁-C₄ alkylene-NR⁷R⁸. In someembodiments, R³ in Formula (I) and Formula (Ia) is —C₂— alkylene-NR⁷R⁸.In some embodiments, R³ in Formula (I) and Formula (Ia) is —C₂—alkylene-NR⁷R⁸. Tn some embodiments, R³ in Formula (I) and Formula (Ia)is —C₃— alkylene-NR⁷R⁸. In some embodiments, R³ in Formula (I) andFormula (Ia) is —C₄— alkylene- NR⁷R⁸. In some embodiments, R³ in Formula(I) and Formula (Ia) is —C₅— alkylene-NR⁷R⁸. In some embodiments, R³ inFormula (I) and Formula (Ia) is —C₀-C₁₀ alkylene-heterocycloalkyl. Insome embodiments, R³ in Formula (I) and Formula (Ia) is —C₁-C₆alkylene-heterocycloalkyl, wherein the heterocycloalkyl comprises 1 to 3nitrogen and 0-2 oxygen. In some embodiments, R³ in Formula (I) andFormula (1a) is —C₁-C₆ alkylene-heterocycloaryl.

In some embodiments, each of R⁷ and R⁸ in Formula (I) and Formula (Ia)is independently hydrogen or substituted or unsubstituted C₁-C₆ alkyl.Tn some embodiments, each of R⁷ and R⁸ is independently hydrogen orsubstituted or unsubstituted C₁-C₃ alkyl. In some embodiments, each ofR⁷ and R⁸ is independently substituted or unsubstituted C₁-C₃ alkyl. Insome embodiments, each of R⁷ and R⁸ is independently —CH₃, —CH₂CH₃,—CH₂CH₂CH₃, or —CH(CH₃)₂. In some embodiments, each of R and R⁸ is CH₃.In some embodiments, each of R⁷ and R⁸ is —CH₂CH₃.

In some embodiments, R⁷ and R⁸ in Formula (I) and Formula (Ia) takentogether with the nitrogen to which they are attached form a substitutedor unsubstituted C₂-C₆ heterocyclyl. In some embodiments, R⁷ and R⁸taken together with the nitrogen to which they are attached form asubstituted or unsubstituted C₂-C₆ heterocycloalkyl. In someembodiments, R⁷ and R⁸ taken together with the nitrogen to which theyare attached form a substituted or unsubstituted 3-7 memberedheterocycloalkyl.

In some embodiments, R³ in Formula (I) and Formula (1a) is

In some embodiments. R³ in Formula (I) and Formula (Ia) is

In some embodiments. R³ in Formula (1) and Formula (1a) is

In some embodiments, Z in Formula (I) and Formula (Ia) is —C(═O)O— or—OC(═O)—. In some embodiments, Z in Formula (1) and Formula (Ia) is—C(═O)NR⁴— or —NR⁴C(═O)—. In some embodiments, Z in Formula (I) andFormula (Ia) is —C(═O)N(CH₃)—, —N(CH₃)C(═O)—, —C(═O)NH—, or —NHC(═O)—.In some embodiments, Z in Formula (I) and Formula (Ia) is —OC(═O)O—,—NR⁴C(═O)O—, —OC(O)NR⁴—, or —NR⁴C(═O)NR⁴—. In some embodiments. Z inFormula (I) and Formula (Ia) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—,—NHC(═O)NH—, —N(CH₃)C(═O)O—, —OC(═O)N(CH₃)—, —N(CH₃)C(═O)N(CH₃)—,—NHC(═O)N(CH₃)— or —N(CH₃)C(═O)NH—. In some embodiments, Y in Formula(I) and Formula (Ia) is —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, or—NHC(═O)NH—.

In some embodiments, R⁵ in Formula (I) and Formula (Ia) is hydrogen orsubstituted or unsubstituted C₁-C₃ alkyl. In some embodiments, R⁵ inFormula (I) and Formula (Ia) is H, —CH₃, —CH—)CH₃, —CH₂CH₂CH₃, or—CH(CH₃)₂. In some embodiments, R⁵ in Formula (I) and Formula (Ia) is H.

In some embodiments, the LNP comprises a plurality of amino lipids. Forexample, the LNP composition can comprise 2, 3, 4, 5, 6.7, 8, 9, 10, ormore amino lipids. For another example, the LNP composition can compriseat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 9, at least 10, or at least 20 amino lipids. For yet anotherexample, the LNP composition can comprise at most 23, at most 4, at most5, at most 6, at most 7, at most 9, at most 10, at most 20, or at most30 amino lipids.

In some embodiments, the LNP composition comprises a first amino lipid.In some embodiments, the LNP composition comprises a first amino lipidand a second amino lipid. In some embodiments, the LNP compositioncomprises a first amino lipid, a second amino lipid, and a third aminolipid. In some embodiments, the LNP composition comprises a first aminolipid, a second amino lipid, a third amino lipid, and a fourth aminolipid. In some embodiments, the LNP composition does not comprise afourth amino lipid. In some embodiments, the LNP composition does notcomprise a third amino lipid. In some embodiments, a molar ratio of thefirst amino lipid to the second amino lipid is from about 0.1 to about10. In some embodiments, a molar ratio of the first amino lipid to thesecond amino lipid is from about 0.20 to about 5. In some embodiments, amolar ratio of the first amino lipid to the second amino lipid is fromabout 0.25 to about 4. In some embodiments, a molar ratio of the firstamino lipid to the second amino lipid is about 0.25, about 0.33, about0.5, about 1, about 2, about 3, or about 4.

In some embodiments, a molar ratio of the first amino lipid:the secondamino lipid:the third amino lipid is about 4:1:1. In some embodiments, amolar ratio of the first amino lipid:the second amino lipid:the thirdamino lipid is about 1:1:1. In some embodiments, a molar ratio of thefirst amino lipid:the second amino lipid: the third amino lipid is about2:1:1. In some embodiments, a molar ratio of the first amino lipid:thesecond amino lipid: the third amino lipid is about 2:2:1. In someembodiments, a molar ratio of the first amino lipid:the second aminolipid:the third amino lipid is about 3:2:1. In some embodiments, a molarratio of the first amino lipid:the second amino lipid: the third aminolipid is about 3:1:1. In some embodiments, a molar ratio of the firstamino lipid:the second amino lipid:the third amino lipid is about 5:1:1.In some embodiments, a molar ratio of the first amino lipid:the secondamino lipid:the third amino lipid is about 3:3:1. In some embodiments, amolar ratio of the first amino lipid:the second amino lipid: the thirdamino lipid is about 4:4:1.

In some embodiments, the LNP composition comprises one or more aminolipids. In some embodiments, the one or more amino lipids comprise fromabout 40 mol % to about 65 mol % of the total lipid present in theparticle. In some embodiments, the one or more amino lipids compriseabout 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44mol %, about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %,about 49 mol %, about 50 mol %, about 51 mol %, about 52 mol %, about 53mol %, about 54 mol %, about 55 mol %, about 56 mol %, about 57 mol %,about 58 mol %, about 59 mol %, about 60 mol %, about 61 mol %, about 62mol %, about 63 mol %, about 64 mol %, or about 65 mol % of the totallipid present in the particle. In some embodiments, the first aminolipid comprises from about 1 mol % to about 99 mol % of the total aminolipids present in the particle. In some embodiments, the first aminolipid comprises from about 16.7 mol % to about 66.7 mol % of the totalamino lipids present in the particle. In some embodiments, the firstamino lipid comprises from about 20 mol % to about 60 mol % of the totalamino lipids present in the particle.

In some embodiments, the amino lipid is an ionizable lipid. An ionizablelipid can comprise one or more ionizable nitrogen atoms. In someembodiments, at least one of the one or more ionizable nitrogen atoms ispositively charged. Tn some embodiments, at least 10 mol %, 20 mol %, 30mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %. 90 mol %, 95mol %, or 99 mol % of the ionizable nitrogen atoms in the LNPcomposition are positively charged. In some embodiments, the amino lipidcomprises a primary amine, a secondary amine, a tertiary amine, animine, an amide, a guanidine moiety, a histidine residue, a lysineresidue, an arginine residue, or any combination thereof. In someembodiments, the amino lipid comprises a primary amine, a secondaryamine, a tertiary amine, a guanidine moiety, or any combination thereof.In some embodiments, the amino lipid comprises a tertiary amine.

In some embodiments, the amino lipid is a cationic lipid. Tn someembodiments, the amino lipid is an ionizable lipid. In some embodiments,the amino lipid comprises one or more nitrogen atoms. In someembodiments, the amino lipid comprises one or more ionizable nitrogenatoms. Exemplary cationic and/or ionizable lipids include, but are notlimited to, 3-(didodecylamino)- N1,N1,4-tri dodecyl-1-piperazineethanamine (KL10),N142-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4—dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC 3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), and(2S)—2-({8-[(33)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).

In some embodiments, an amino lipid described herein can take the formof a salt, such as a pharmaceutically acceptable salt. Allpharmaceutically acceptable salts of the amino lipid are encompassed bythis disclosure. As used herein, amino lipid also includes itspharmaceutically acceptable salts, and its diastereomeric, enantiomeric,and epimeric forms.

In some embodiments, an amino lipid described herein, possesses one ormore stereocenters and each stereocenter exists independently in eitherthe R or S configuration. The lipids presented herein include alldiastereomeric, enantiomeric, and epimeric forms as well as theappropriate mixtures thereof. The lipids provided herein include allcis. trans, syn, anti, entgegen (E), and zusammen (Z) isomers as well asthe appropriate mixtures thereof. Tn certain embodiments, lipidsdescribed herein are prepared as their individual stereoisomers byreacting a racemic mixture of the compound with an optically activeresolving agent to form a pair of diastereoisomeric compounds/salts,separating the diastereomers and recovering the optically pureenantiomers. In some embodiments, resolution of enantiomers is carriedout using covalent diastereomeric derivatives of the compounds describedherein. In another embodiment, diastereomers are separated byseparation/resolution techniques based upon differences in solubility.In other embodiments, separation of stereoisomers is performed bychromatography or by the forming diastereomeric salts and separation byrecrystallization, or chromatography, or any combination thereof. JeanJacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates andResolutions”, John Wiley and Sons, Inc., 1981. In one aspect,stereoisomers are obtained by stereoselective synthesis.

In some embodiments, the lipids such as the amino lipids are substitutedbased on the structures disclosed herein. In some embodiments, thelipids such as the amino lipids are unsubstituted. In anotherembodiment, the lipids described herein are labeled isotopically (e.g.with a radioisotope) or by another other means, including, but notlimited to, the use of chromophores or fluorescent moieties,bioluminescent labels, or chemiluminescent labels.

Lipids described herein include isotopically-labeled compounds, whichare identical to those recited in the various formulae and structurespresented herein, but for the fact that one or more atoms are replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present lipids include isotopes ofhydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, suchas, for example, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S ¹⁸F, ³⁶Cl. In oneaspect, isotopically-labeled lipids described herein, for example thoseinto which radioactive isotopes such as ³H and ¹⁴C are incorporated, areuseful in drug and/or substrate tissue distribution assays. In oneaspect, substitution with isotopes such as deuterium affords certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements.

In some embodiments, the asymmetric carbon atom of the amino lipid ispresent in enantiomerically enriched form. In certain embodiments, theasymmetric carbon atom of the amino lipid has at least 50% enantiomericexcess, at least 60% enantiomeric excess, at least 70% enantiomericexcess, at least 80% enantiomeric excess, at least 90% enantiomericexcess, at least 95% enantiomeric excess, or at least 99% enantiomericexcess in the (S)— or (R)-configuration.

In some embodiments, the disclosed amino lipids can be converted toN-oxides. In some embodiments, N-oxides are formed by a treatment withan oxidizing agent (e.g., 3-chloroperoxybenzoic acid and/or hydrogenperoxides). Accordingly, disclosed herein are N-oxide compounds of thedescribed amino lipids, when allowed by valency and structure, which canbe denated as N O or N⁺—O⁻. In some embodiments, the nitrogen in thecompounds of the disclosure can be converted to N-hydroxy or N-alkoxy.For example, N-hydroxy compounds can be prepared by oxidation of theparent amine by an oxidizing agent such as ra-CPBA. All shown andclaimed nitrogencontaining compounds are also considered. Accordingly,also disclosed herein are N-hydroxy and Nal koxy (e.g., N—OR, wherein Ris substituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle)derivatives of the described amino lipids.

PEG-Lipid

As used herein, a “PEG lipid” or “PEG-lipid” refers to a lipidcomprising a polyethylene glycol component.

In some embodiments, the described LNP composition comprises aPEG-lipid. In some embodiments, the described LNP composition comprisestwo or more PEG-lipids. Exemplary PEG—lipids include, but are notlimited to, the lipids. Exemplary PEG-lipids also include, but are notlimited to, PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG—modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, the one or more PEG-lipids can comprisePEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid, ora combination thereof. In some embodiments, PEG moiety is an optionallysubstituted linear or branched polymer of ethylene glycol or ethyleneoxide. In some embodiments, the PEG moiety is substituted, e.g., by oneor more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In someembodiments, the PEG moiety includes PEG copolymer such asPEG-polyurethane or PEG-polypropylene (see, e.g., j. Milton Harris,Poly(ethylene glycol) chemistry: biotechnical and biomedicalapplications (1992)). In some embodiments, the PEG moiety does notinclude PEG copolymers, e.g., it may be a PEG monopolymer. ExemplaryPEG-lipids include, but are not limited to, PEG-dilauroylglycerol,PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol,PEG-distearoylgiycerol (PEG-DSPE), PEG-dipalmitoylglycerol,PEG-disteiylglycerol, PEG-dilawylglycamide, PEG-dimyristylglycamide,PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, andPEG-DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]).

In some embodiments, a PEG-lipid is a PEG-lipid conjugate, for example,PEG coupled to dialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupledto diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled tocholesterol, PEG coupled to phosphatidylethanolamines, and PEGconjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613), cationicPEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g. POZ-DAAconjugates; see, e.g., WO 2010/006282), polyamide oligomers (e.g.,ATTA-lipid conjugates), and mixtures thereof.

A PEG-lipid can comprise one or more ethylene glycol units, for example,at least 1, at least 2, at least 5, at least 10, at least 20, at least30, at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, at least 100, at least 120, or at least 150 ethylene glycolunits. In some embodiments, a number average molecular weight of thePEG-lipids is from about 200 Da to about 5000 Da. In some embodiments, anumber average molecular weight of the PEG-lipids is from about 500 Dato about 3000 Da. In some embodiments, a number average molecular weightof the PEG-lipids is from about 750 Da to about 2500 Da. In someembodiments, a number average molecular weight of the PEG- lipids isfrom about 750 Da to about 2500 Da. In some embodiments, a numberaverage molecular weight of the PEG-lipids is about 500 Da, about 750Da, about 1000 Da, about 1250 Da, about 1500 Da, about 1750 Da, or about2000 Da. In some embodiments, a polydispersity index (PD1) of the one ormore PEG-lipids is smaller than 2. In some embodiments, a PDI of the oneor more PEG-lipids is at most 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In someembodiments, a PDI of the one or more PEG-lipids is at least 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, or 3.0.

In some embodiments, the PEG-lipid comprises from about 0.1 mol % toabout 10 mol % of the total lipid present in the particle. In someembodiments, the PEG-lipid comprises from about 0.1 mol % to about 6 mol% of the total lipid present in the particle. In some embodiments, thePEG-lipid comprises from about 0.5 mol % to about 5 mol % of the totallipid present in the particle. In some embodiments, the PEG-lipidcomprises from about 1 mol % to about 3 mol % of the total lipid presentin the particle. In some embodiments, the PEG-lipid comprises about 2.0mol % to about 2.5 mol % of the total lipid present in the particle. Insome embodiments, the PEG-lipid comprises about 1 mol %, about 1.1 mol%, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %,about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %,about 2.0 mol %, about 2.1 mol %, about 2.2 mol %, about 2.3 mol %,about 2.4 mol %, about 2.5 mol %, about 2.6 mol %, about 2.7 mol %,about 2.8 mol %, about 2.9 mol %, or about 3.0 mol % of the total lipidpresent in the particle.

In some embodiments, the LNP composition comprises a plurality ofPEG-lipids, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredistinct PEG-lipids.

Phospholipid

As used herein, a “phospholipid” refers to a lipid that includes aphosphate moiety and one or more carbon chains, such as unsaturatedfatty acid chains. A phospholipid may include one or more multiple(e.g., double or triple) bonds. In some embodiments, a phospholipid mayfacilitate fusion to a membrane. For example, a cationic phospholipidmay interact with one or more negatively charged phospholipids of amembrane (e.g., a cellular or intracellular membrane). Fusion of aphospholipid to a membrane may allow one or more elements of an LNP topass through the membrane, i.e., delivery of the one or more elements toa cell.

In some embodiments, the described LNP composition comprises aphospholipid. In some embodiments, the phospholipid comprises a lipidselected from the group consisting of: phosphatidylcholine (PC),phosphatidylethanolamine amine, glycerophospholipid,sphingophospholipids, Guriserohosuhono, sphingolipids phosphono lipids,natural lecithins, and hydrogenated phospholipid. In some embodiments,the phospholipid comprises a phosphatidylcholine. Exemplaryphosphatidylcholines include, but are not limited to, soybeanphosphatidylcholine, egg yolk phosphatidylcholine (EPC),distearoylphosphatidylcholine,1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoylphosphatidylcholine, dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), dimyristoylphosphatidylcholine (DMPC), and dioleoyl phosphatidylcholine (DOPC). Incertain specific embodiments, the phospholipid is DSPC.

In some embodiments, the phospholipid comprises aphosphatidylethanolamine amine. In some embodiments, thephosphatidylethanolamine amine is distearoyl phosphatidylethanolamine(DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dimyristoylphosphoethanolamine (DMPE), 16-0-Monome Le PE, 16-0-dimethyl PE,18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine (POPE), or1-stearoyl-2- oleoyl-phosphatidyl ethanolamine (SOPE). In someembodiments, the phospholipid comprises a glycerophospholipid. In someembodiments, the glycerophospholipid is plasmalogen, phosphatidate, orphosphatidylcholine. In some embodiments, the glycerophospholipid isphosphatidylserine, phosphatidic acid, phosphatidylglycerol,phosphatidylinositol, palmitoyl oleoyl phosphatidylglycerol (POPG), orlysophosphatidylcholine. In some embodiments, the phospholipid comprisesa sphingophospholipid. In some embodiments, the sphingophospholipid issphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol,or ceramide phosphoglycerophosphoric acid. In some embodiments, thephospholipid comprises a natural lecithin. In some embodiments, thenatural lecithin is egg yolk lecithin or soybean lecithin. Tn someembodiments, the phospholipid comprises a hydrogenated phospholipid. Insome embodiments, the hydrogenated phospholipid is hydrogenated soybeanphosphatidylcholine. In some embodiments, the phospholipid is selectedfrom the group consisting of: phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.

In some embodiments, the phospholipid comprises a lipid selected from:1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 2-Oleoyl-1-pahnitoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di docosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diaracliidonoyl-sn-gly cero-3- phosphoethanol amine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diol eoy I -sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), andsphingomyelin.

A phospholipid can comprise a phospholipid moiety and one or more fattyacid moieties. A phospholipid moiety can comprise phosphatidyl choline,phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine,phosphatidic acid, 2-lysophosphatidyl choline, or a sphingomyelin. Afatty acid moiety can comprise lauric acid, myristic acid, myristoleicacid, palmitic acid, palmitoleic acid, stearic acid, oleic acid,linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid,arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid,docosapentaenoic acid, or docosahexaenoic acid. In some specificembodiments, a phospholipid can be functionalized with or cross-linkedto one or more alkynes, which may undergo a copper-catalyzedcycloaddition upon exposure to an azide.

In some embodiments, the LNP composition comprises a plurality ofphospholipids, for example, at least 2, 3, 4, 5, or more distinctphospholipids. In some embodiments, the phospholipid comprises from 1mol % to 20 mol % of the total lipid present in the particle. In someembodiments, the phospholipid comprises from about 5 mol % to about 15mol % of the total lipid present in the particle. In some embodiments,the phospholipid comprises from about 8 mol % to about 12 mol % of thetotal lipid present in the particle. In some embodiments, thephospholipid comprises from about 9 mol %, 10 mol %, or 11 mol % of thetotal lipid present in the particle.

Cholesterol

In some embodiments, the LNP composition comprises a cholesterol or aderivative thereof. In some embodiments, the LNP composition comprises astructural lipid. The structural lipid can be selected from steroid,sterol, alkyl resoreinol, cholesterol or derivative thereof, fecosterol,sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,tomatidine, tomatine, ursolic acid, alphatocopherol, and a combinationthereof. In some embodiments, the structural lipid is a corticosteroidsuch as prednisolone, dexamethasone, prednisone, and hydrocortisone. Insome embodiments, the cholesterol or derivative thereof is cholesterol,5- heptadecylresorcinol, or cholesterol hemisuccinate. Tn someembodiments, the cholesterol or derivative thereof is cholesterol.

In some embodiments, the cholesterol or derivative thereof is acholesterol derivative. In some embodiments, the cholesterol derivativeis a polar cholesterol analogue. In some embodiments, the polarcholesterol analogue is 5a-cholestanol, 513-coprostanol,cholesteryl-(2′-hydroxy)-ethyl ether, cholestely1-(4′-hydroxy)-butylether, or 6-ketocholestanol. In some embodiments, the polar cholesterolanalogue is cholesteryl-(4′-hydroxy)-butyl ether. In some embodiments,the cholesterol derivative is a non-polar cholesterol analogue. In someembodiments, the non-polar cholesterol analogue is 5acholestane,cholestenone, 5α-cholestanone, 5β-cholestanone, or cholestetyldecanoate.

In some embodiments, the cholesterol or the derivative thereof comprisesfrom 20 mol % to 50 mol % of the total lipid present in the particle. Insome embodiments, the cholesterol or the derivative thereof comprisesabout 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24mol %, about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %,about 29 mol %, about 30 mol %, about 31 mol %, about 32 mol %, about 33mol %, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %,about 38 mol %, about 39 mol %, about 40 mol %, about 41 mol %, about 42mol %, about 43 mol %, about 44 mol %. about 45 mol %. about 46 mol %,about 47 mol %, about 48 mol %, or about 50 mol % of the total lipidpresent in the particle.

Phosphate Charge Neutralizer

In some embodiments, the LNP described herein comprises a phosphatecharge neutralizer. In some embodiments, the phosphate chargeneutralizer comprises arginine, asparagine, glutamine, lysine,histidine, cationic dendrimers, polyamines, or a combination thereof. Insome embodiments, the phosphate charge neutralizer comprises one or morenitrogen atoms. In some embodiments, the phosphate charge neutralizercomprises a polyamine. In some embodiments, the polyamine is 1,3-propanediamine, spermine, spermidine, Norspermidine,Tris(2-aminoethy)amine, Cyclen, 1,4,7- Triazacyclononane,1,1,1-Tris(aminomethyl)ethane, Diethylenetriamine, Triethylenetetramine,or a combination thereof. In some embodiments, the polyamine is1,3-propanediamine, 1,4-butanediamine, spermine, spermidine, or acombination thereof. In some embodiments, the N/P ratio for thephosphate charge neutralizer is from 0.01 to 10. In some embodiments,the N/P ratio for the phosphate charge neutralizer is from about 0.05 toabout 2. In some embodiments, the N/P ratio for the phosphate chargeneutralizer is from about 0.1 to about 1. In some embodiments, the N/Pratio for the phosphate charge neutralizer is about 0.1, about 0.2,about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about0.9, or about 1. In some embodiments, the NIP ratio for the phosphatecharge neutralizer is about 0.25, 0.5, or 0.75.

Antioxidants

In some embodiments, the LNP described herein comprises one or moreantioxidants. In some embodiments, the one or more antioxidants functionto reduce a degradation of the cationic lipids, the payload, or both. Insome embodiments, the one or more antioxidants comprise a hydrophilicantioxidant. In some embodiments, the one or more antioxidants is achelating agent such as ethylenediaminetetraacefic acid (EDTA) andcitrate. In some embodiments, the one or more antioxidants is EDTA. Insome embodiments, the one or more antioxidants comprise a lipophilicantioxidant. In some embodiments, the lipophilic antioxidant comprises avitamin E isomer or a polyphenol. In some embodiments, the one or moreantioxidants are present in the LNP composition at a concentration of atleast 1 mM, at least 10 mM, at least 20 mM, at least 50 mM, or at least100 mM. In some embodiments, the one or more antioxidants are present inthe particle at a concentration of about 20 mM.

Payload

The LNPs described herein can be designed to deliver a payload, such asa therapeutic agent, or a target of interest. In some embodiments, anLNP described herein encloses one or more components of a base editorsystem as described herein. For example, a LNP may enclose one or moreof a guide RNA, a nucleic acid encoding the guide RNA, a vector encodingthe guide RNA, a base editor fusion protein, a nucleic acid encoding thebase editor fusion protein, a programmable DNA binding domain, a nucleicacid encoding the programmable DNA binding domain, a deaminase, anucleic acid encoding the deaminase, or all or any combination thereof.In some embodiments, the nucleic acid is a DNA. In some embodiments, thenucleic acid is a RNA, for example, a mRNA.

Additional exemplary therapeutic agents include, but are not limited to,antibodies (e.g., monoclonal, chimeric, humanized, nanobodies, andfragments thereof etc.), cholesterol, hormones, peptides, proteins,chemotherapeutics and other types of antineoplastic agents, lowmolecular weight drugs, vitamins, co-factors, nucleosides, nucleotides,oligonucleotides, enzymatic nucleic acids, antisense nucleic acids,triplex forming oligonucleotides, antisense DNA or RNA compositions,chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys andanalogs thereof, plasmids and other types of expression vectors, andsmall nucleic acid molecules, RNAi agents, short interfering nucleicacid (siNA), messenger ribonucleic acid (messenger RNA, mRNA), shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), alocked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threosenucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internallysegmented interfering RNA), aiRNA (asymetrical interfering RNA), andsiRNA with 1, 2 or more mismatches between the sense and anti-sensestrand to relevant cells and/or tissues, such as in a cell culture,subject or organism. Therapeutic agents can be purified or partiallypurified, and can be naturally occurring or synthetic, or chemicallymodified. In some embodiments, the therapeutic agent is an RNAi agent,short interfering nucleic acid (siNA), short interfering RNA (siRNA),double- stranded RNA (dsRNA), micro-RNA (miRNA), or a short hairpin RNA(shRNA) molecule. In some embodiments, the therapeutic agent is an mRNA.

In some embodiments, the payload comprises one or more nucleic acid(s)(i.e., one or more nucleic acid molecular entities). In someembodiments, the nucleic acid is a single-stranded nucleic acid. In someembodiments, single-stranded nucleic acid is a DNA. In some embodiments,single- stranded nucleic acid is an RNA. In some embodiments, thenucleic acid is a double-stranded nucleic acid. In some embodiments, thedouble-stranded nucleic acid is a DNA. In some embodiments, thedouble-stranded nucleic acid is an RNA. In some embodiments, thedouble-stranded nucleic acid is a DNA-RNA hybrid. In some embodiments,the nucleic acid is a messenger RNA (mRNA), a microRNA, an asymmetricalinterfering RNA (aiRNA), a small hairpin RNA (shRNA), or a Dicer-Substrate dsRNA.

Other Lipids

In some embodiments, the disclosed LNP compositions comprise a helperlipid. In some embodiments, the disclosed LNP compositions comprise aneutral lipid. In some embodiments, the disclosed LNP compositionscomprise a stealth lipid. In some embodiments, the disclosed LNPcompositions comprises additional lipids.

As used herein, neutral lipids suitable for use in a lipid compositionof the disclosure include, for example, a variety of neutral, unchargedor zwitterionic lipids. Examples of neutral phospholipids suitable foruse in the present disclosure include, but are not limited to,5-heptadecylbenzene-1,3-diol (resorcinol),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), phosphocholine (DOPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), phosphatidylcholine(PLPC), 1,2-distearoyl- sn-glycero-3-phosphocholine (DAPC),phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC),dilauryloylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine(PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC),1,2-diarachidoylsn- glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),1,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC),2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC),lysophosphatidyl choline, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE), dilinoleoylphosphatidylcholinedistearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine(DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE),lysophosphatidylethanolamine and combinations thereof. In someembodiments, the neutral phospholipid is selected from the groupconsisting of DSPC and dimyristoyl phosphatidyl ethanolamine (DMPE). Insome embodiments, the neutral phospholipid is DSPC. Neutral lipids canfunction to stabilize and improve processing of the LNPs.

Helper lipids can refer to lipids that enhance transfection (e.g.transfection of the nanoparticle (LNP) comprising the composition asprovided herein, including the biologically active agent). The mechanismby which the helper lipid enhances transfection includes enhancingparticle stability. In some embodiments, the helper lipid enhancesmembrane fusogenicity. Helper lipids can include steroids, sterols, andalkyl resorcinols. Helper lipids suitable for use in the presentdisclosure can include, but are not limited to, cholesterol,5-heptadecylresorcinol, and cholesterol hemisuccinate. In someembodiments, the helper lipid is cholesterol. In some embodiments, thehelper lipid is be cholesterol hemisuccinate.

Stealth lipids can refer to lipids that alter the length of time thenanoparticles can exist in vivo (e.g., in the blood). Stealth lipids canassist in the formulation process by, for example, reducing particleaggregation and controlling particle size. Stealth lipids used hereinmay modulate pharmacokinetic properties of the LNP. Stealth lipidssuitable for use in a lipid composition of the disclosure can include,but are not limited to, stealth lipids having a hydrophilic head grouplinked to a lipid moiety. Stealth lipids suitable for use in a lipidcomposition of the present disclosure and information about thebiochemistry of such lipids can be found in Romberg et al,Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and I-Toekstraet al, Biochimica et Biophysica Acta 1660 (2004) 41-52. Additionalsuitable PEG lipids are disclosed, e.g., in WO 2006/007712.

In some embodiments, the stealth lipid is a PEG-lipid. In oneembodiment, the hydrophilic head group of stealth lipid comprises apolymer moiety selected from polymers based on PEG (sometimes referredto as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol),poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids and polyN-(2-hydroxypropyl)methacrylamide]. Stealth lipids can comprise a lipidmoiety. In some embodiments, the lipid moiety of the stealth lipid maybe derived from diacylglycerol or diacylglycamide, including thosecomprising a dialkylglycerol or dialkylglycamide group having alkylchain length independently comprising from about C4 to about C40saturated or unsaturated carbon atoms, wherein the chain may compriseone or more functional groups such as, for example, an amide or ester.The dialkylglycerol or dialkylglycamide group can further comprise oneor more substituted alkyl groups.

The structures and properties of helper lipids, neutral lipids, stealthlipids, and/or other lipids are further described in W02017173054A1,W02019067999A1, US20180290965A1, US20180147298A1, US20160375134A1, U.S.Pat. Nos. 8,236,770, 8,021,686, 8,236,770B2, U.S. Pat. No. 7,371,404B2,U.S. Pat. No. 7,780,983B2, U.S. Pat. No. 7,858,117B2, US20180200186A1,US20070087045A1, W02018119514A1, and W02019067992A1, all of which arehereby incorporated by reference in their entirety.

LNP Formulations

The LNPs described herein can be designed for one or more specificapplications or targets. The elements of a nanoparticle (LNP)composition or a composition can be selected based on a particularapplication or target, and/or based on the efficacy, toxicity, expense,ease of use, and availability. Similarly, the particular formulation ofa nanoparticle composition is a composition comprising one or moredescribed lipids. may be selected for the particular application ortarget. Suitable phosphate charge neutralizers to be used informulations include, but are not limited to, Spermidine and1,3-propanediamine.

The described LNP formulations can be designed for one or more specificapplications or targets. For example, a nanoparticle composition may bedesigned to deliver a therapeutic agent such as an RNA to a particularcell, tissue, organ, or system or group thereof in a mammal's body.Physiochemical properties of nanoparticle compositions may be altered inorder to increase selectivity for particular bodily targets. Forinstance, particle sizes may be adjusted based on the fenestration sizesof different organs. The therapeutic agent included in a nanoparticlecomposition may also be selected based on the desired delivery target ortargets. For example, a therapeutic agent may be selected for aparticular indication, condition, disease, or disorder and/or fordelivery to a particular cell, tissue, organ, or system or group thereof(e.g., localized or specific delivery). In certain embodiments, ananoparticle composition may include an mRNA encoding a polypeptide ofinterest capable of being translated within a cell to produce thepolypeptide of interest. Such a composition may be designed to bespecifically delivered to a particular organ.

The amount of a therapeutic agent in an LNP composition may depend onthe size, composition, desired target and/or application, or otherproperties of the nanoparticle composition. For example, the amount ofan RNA comprised in a nanoparticle composition may depend on the size,sequence, and other characteristics of the RNA. The relative amounts ofa therapeutic agent and other elements (e.g., lipids) in a nanoparticlecomposition may also vary. In some embodiments, the wt/wt ratio of thelipid component to a therapeutic agent in a nanoparticle composition maybe from about 5:1 to about 60:1, such as about 5:1. 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1,30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt/wt ratio ofthe lipid component to a therapeutic agent may be from about 10:1 toabout 40:1. In certain embodiments, the wt/wt ratio is about 20:1. Theamount of a therapeutic agent in a nanoparticle composition can bemeasured using absorption spectroscopy (e.g., ultraviolet-visiblespectroscopy).

In some embodiments, an LNP composition comprises one or more nucleicacids such as RNAs. In some embodiments, the one or more RNAs, lipids,and amounts thereof may be selected to provide a specific NIP ratio. TheNIP ratio can be selected from about 1 to about 30. The N/P ratio can beselected from about 2 to about 10. In some embodiments, the NIP ratio isfrom about 0.1 to about 50. In some embodiments, the N/P ratio is fromabout 2 to about 8. hi some embodiments, the NIP ratio is from about 2to about 15, from about 2 to about 10, from about 2 to about 8, fromabout 2 to about 6, from about 3 to about 15, from about 3 to about 10,from about 3 to about 8, from about 3 to about 6, from about 4 to about15, from about 4 to about 10, from about 4 to about 8, or from about 4to about 6. hi some embodiments, the N/P ratio is about 2, about 2.5,about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, orabout 6.5. In some embodiments, the NIP ratio is from about 4 to about6. In some embodiments, the NIP ratio is about 4, about 4.5, about 5,about 5.5, or about 6.

In some embodiments, the LNPs are formed with an average encapsulationefficiency ranging from about 50% to about 70%, from about 70% to about90%, or from about 90% to about 100%. In some embodiments, the LNPs areformed with an average encapsulation efficiency ranging from about 75%to about 95%.

In another aspect, provided herein is a lipid nanoparticle (LNP)comprising the composition as provided herein. As used herein, a lipidnanoparticle (LNP) composition or a nanoparticle composition is acomposition comprising one or more described lipids. LNP compositionsare typically sized on the order of micrometers or smaller and mayinclude a lipid bilayer. Nanoparticle compositions encompass lipidnanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.In some embodiments, a LNP refers to any particle that has a diameter ofless than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm,or 25 nm. In some embodiments, a nanoparticle may range in size from1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60nm. In some embodiments, a liposome having a lipid bilayer with adiameter of 500 rim or less. In some embodiments, the LNPs describedherein can have a mean diameter of from about 1 nm to about 2500 nm,from about 10 nm to about 1500 nm, from about 20 nm to about 1000 nm,from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 rim to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm- or from about70 nm to about 80 nm. The LNPs described herein can have a mean diameterof about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm,75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, or greater. The LNPsdescribed herein can be substantially non-toxic.

In some embodiments, an LNP may be made from cationic, anionic, orneutral lipids. In some embodiments, an LNP may comprise neutral lipids,such as the fusogenic phospholipid1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membranecomponent cholesterol, as helper lipids to enhance transfection activityand nanoparticle stability. In some embodiments, an LNP may comprisehydrophobic lipids, hydrophilic lipids, or both hydrophobic andhydrophilic lipids. Any lipid or combination of lipids that are known inthe art can be used to produce an LNP. Examples of lipids used toproduce LNPs include, but are not limited to DOTMA(N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOSPA(N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)-1-propaniminiumpentahydrochloride), DOTAP (1,2-Dioleoyl-3-trimethylammonium propane),DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-1-propanaminiumbromide),DC-cholesterol(3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol),DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE(2-Bis(dimethylphosphino)ethane)-polyethylene glycol (PEG). Examples ofcationic lipids include, but are not limited to, 98N12-5, C12-200,DLin-KC2-DMA (KC2), DLin-MC3 -DMA (MC3), XTC, MD1, and 7C1. Examples ofneutral lipids include, but are not limited to, DPSC, DPPC(Dipalmitoylphosphatidylcholine), POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE, and SM(sphingomyelin). Examples of PEG-modified lipids include, but are notlimited to, PEG-DMG (Dimyristoyl glycerol), PEG-CerC14, and PEG-CerC20.In some embodiments, the lipids may be combined in any number of molarratios to produce a LNP. In some embodiments, the polynucleotide may becombined with lipid(s) in a wide range of molar ratios to produce anLNP.

The term “substituted”, unless otherwise indicated, refers to thereplacement of one or more hydrogen radicals in a given structure withthe radical of a specified substituent including, but not limited to:halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio,oxo, thioxy, arylthio, alkylthioalkyl, arylthioallcyl, alkylsulfonyl,alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy,aminocarbonyl, alkylaminocarbonyl, aiylaminocarbonyl, alkoxycarbonyl,aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro,alkylamino, arylamino, alkylaminoalkyl, aiylaminoalkyl, aminoalkylamino,hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl,aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonicacid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and analiphatic group. It is understood that the substituent may be furthersubstituted. Exemplary• substituents include amino, alkylamino, and thelike.

As used herein, the term “substituent” means positional variables on theatoms of a core molecule that are substituted at a designated atomposition, replacing one or more hydrogens on the designated atom,provided that the designated atom's normal valency is not exceeded, andthat the substitution results in a stable compound. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds. A person of ordinal), skill in the artshould note that any carbon as well as heteroatom with valences thatappear to be unsatisfied as described or shown herein is assumed to havea sufficient number of hydrogen atom(s) to satisfy the valencesdescribed or shown. In certain instances one or more substituents havinga double bond (e.g., “oxo” or “═O”) as the point of attachment may bedescribed, shown or listed herein within a substituent group, whereinthe structure may only show a single bond as the point of attachment tothe core structure of Formula (I). A person of ordinary skill in the artwould understand that, while only a single bond is shown, a double bondis intended for those substituents.

The term “alkyl” refers to a straight or branched hydrocarbon chainradical, having from one to twenty carbon atoms, and which is attachedto the rest of the molecule by a single bond. An alkyl comprising up to10 carbon atoms is referred to as a C₁-C₁₀ alkyl, likewise, for example,an alkyl comprising up to 6 carbon atoms is a C₁-C₆ alkyl. Alkyls (andother moieties defined herein) comprising other numbers of carbon atomsare represented similarly. Alkyl groups include, but are not limited to,C₁-C₁₀ alkyl, C₁-C₉ alkyl, C₁-C₈ alkyl. C₁-C₇ alkyl, C₁-C₆ alkyl, C₁-C₅alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl, C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈ alkyland C₄-C₈ alkyl. Representative alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl,i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, thealkyl is methyl or ethyl. In some embodiments, the alkyl is —CH(CH₃)₂ or—C(CH₃)₃. Unless stated otherwise specifically in the specification, analkyl group may be optionally substituted as described below. “Alkylene”or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group.In some embodiments, the alkylene is —CI-12—, —CH₂CH₂—, or —CH₂CH₂CH₂—.In some embodiments, the alkylene is —CH₂—. In some embodiments, thealkylene is —CH₂CH₂—. In some embodiments, the alkylene is —CH₂CH₂CH₂—.

The term “alkenyl” refers to a type of alkyl group in which at least onecarbon-carbon double bond is present. In one embodiment, an alkenylgroup has the formula —C(R)═CR², wherein R refers to the remainingportions of the alkenyl group, which may be the same or different. Insome embodiments, R is H or an alkyl. In some embodiments, an alkenyl isselected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl,pentenyl, pentadienyl, and the like. Non-limiting examples of an alkenylgroup include —CH═CH₂, —C(CH₃)═CH₂, —CH═CHCH₃, —C(CH₃)═CHCH₃, and—CH₂CH═CH₂.

The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromaticradical, wherein each of the atoms forming the ring (i.e. skeletalatoms) is a carbon atom. In some embodiments, cycloalkyls are saturatedor partially unsaturated. In some embodiments, cycloalkyls arespirocyclic or bridged compounds. In some embodiments, cycloalkyls arefused with an aromatic ring (in which case the cycloalkyl is bondedthrough a non-aromatic ring carbon atom). Cycloalkyl groups includegroups having from 3 to 10 ring atoms. Representative cycloalkylsinclude, but are not limited to, cycloalkyls having from three to tencarbon atoms, from three to eight carbon atoms, from three to six carbonatoms, or from three to five carbon atoms. Monocyclic cycloalkylradicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, themonocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl. In some embodiments, the monocyclic cycloalkyl iscyclopentenyl or cyclohexenyl. In some embodiments, the monocycliccycloalkyl is cyclopenteny 1. Polycyclic radicals include, for example,adamantyl, 1,2- dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetrainyl,decalinyl, 3,4-dihydronaphthalenyl-.1 (2H)— one. spiro[2.2]pentyl,norbomyl and bicycle[1.1.1]pentyl. Unless otherwise stated specificallyin the specification, a cycloalkyl group may be optionally substituted.Depending on the structure, a cycloalkyl group can be monovalent ordivalent (i.e., a cycloalkylene group).

The term “heterocycle” or “heterocyclic” refers to heteroaromatic rings(also known as heteroaryls) and heterocycloallcyl rings (also known asheteroalicyclic groups) that includes at least one heteroatom selectedfrom nitrogen, oxygen and sulfur, wherein each heterocyclic group hasfrom 3 to 12 atoms in its ring system, and with the proviso that anyring does not contain two adjacent O or S atoms. A “heterocyclyl” is aunivalent group formed by removing a hydrogen atom from any ring atomsof a heterocyclic compound. In some embodiments, heterocycles aremonocyclic, bicyclic, polycyclic, spirocyclic or bridged compounds.Non-aromatic heterocyclic groups (also known as heterocycloalkyls)include rings having 3 to 12 atoms in its ring system and aromaticheterocyclic groups include rings having 5 to 12 atoms in its ringsystem. The heterocyclic groups include benzofused ring systems.Examples of non-aromatic heterocyclic groups are pyrrolidinyl,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl,tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl,morpholinyl. thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl,azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl,oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl,pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4Hpyranyl,dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl,dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl,imidazolinyl, imidazoli diny I, 3-az.abicy c1 o[3. 1.0]hexany 1,3-azabicyclo[4.1.0]heptanyl, 3 h-indolyl, indolin-2-onyl,isoindolin-1-onyl, isoindoline-1,3-dionyl, 3,4-dihydroisoquinolin-1(2H)-onyl, 3,4-dihydroquinolin-2(1H)-onyl,isoindoline-1,3-dithionyl, benzo[d]oxazol-2(3H)-onyl,1H-benzo[d]imidazol-2(3H)-onyl, benzo[d]thiazol-2(3H)-onyl, andquinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl,imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,futyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furaz.anyl, benzofuraz.anyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, andfuropyridinyl. The foregoing groups are either C-attached (or Clinked)or N-attached where such is possible. For instance, a group derived frompyrrole includes both pyrrol-1-yl (N-attached) or pyrrol-3-yl(C-attached). Further, a group derived from imidazole includesimidazol-1-yl or imidazol-3-yl (both N-attached) or imidazol-2-yl,imidazol-4-yl or imidazol-5-yl (all C-attached). The heterocyclic groupsinclude benzo-fused ring systems. Non-aromatic heterocycles areoptionally substituted with one or two oxo (═O) moieties, such aspyrrolidin-2-one. In some embodiments, at least one of the two rings ofa bicyclic heterocycle is aromatic. In some embodiments, both rings of abicyclic heterocycle are aromatic.

The term “heterocycloalkyl” refers to a cycloalkyl group that includesat least one heteroatom selected from nitrogen, oxygen, and sulfur.Unless stated otherwise specifically in the specification, theheterocycloalkyl radical may be a monocyclic, or bicyclic ring system,which may include fused (when fused with an aryl or a heterowyl ring,the heterocycloalkyl is bonded through a non-aromatic ring atom) orbridged ring systems. The nitrogen, carbon or sulfur atoms in theheterocyclyl radical may be optionally oxidized. The nitrogen atom maybe optionally quaternized. The heterocycloalkyl radical is partially orfully saturated. Examples of heterocycloalkyl radicals include, but arenot limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl,tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl,1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes allring forms of carbohydrates, including but not limited tomonosaccharides, disaccharides and oligosaccharides. Unless otherwisenoted, heterocycloalkyls have from 2 to 12 carbons in the ring. In someembodiments, heterocycloalkyls have from 2 to 10 carbons in the ring. Insome embodiments, heterocycloalkyls have from 2 to 10 carbons in thering and 1 or 2 N atoms. In some embodiments, heterocycloalkyls havefrom 2 to 10 carbons in the ring and 3 or 4 N atoms. In someembodiments, heterocycloalkyls have from 2 to 12 carbons, 0-2 N atoms,0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring. In someembodiments, heterocycloalkyls have from 2 to 12 carbons, 1-3 N atoms,0-1 O atoms, and 0-1 S atoms in the ring. It is understood that whenreferring to the number of carbon atoms in a heterocycloalkyl, thenumber of carbon atoms in the heterocycloalkyl is not the same as thetotal number of atoms (including the heteroatoms) that make up theheterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring).Unless stated otherwise specifically in the specification, aheterocycloalkyl group may be optionally substituted. As used herein,the term “teterocycloalkylene” can refer to a divalent heterocycloalkylgroup.

The term “heteroaryl” refers to an aryl group that includes one or morering heteroatoms selected from nitrogen, oxygen and sulfur. Theheteroaryl is monocyclic or bicyclic. Illustrative examples ofmonocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl,pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl,thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl,oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran,benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,1,8-naphthyridine, and pteridine. Illustrative examples of monocyclicheteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl,triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl,thiadiazolyl, and furazanyl. Illustrative examples of bicyclicheteroaryls include indolizine, indole, benzofuran, benzothiophene,indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline,cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, andpteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl,pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furyl. In someembodiments, a heteroaryl contains 0-6 N atoms in the ring. In someembodiments, a heteroaryl contains 1-4 N atoms in the ring. In someembodiments, a heteroaryl contains 4-6 N atoms in the ring. In someembodiments, a heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 Patoms, and 0-1 S atoms in the ring. In some embodiments, a heteroarylcontains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In someembodiments, heteroaryl is a C₁-C₉ heteroaryl. In some embodiments,monocyclic heteroaryl is a C₁-C₅ heteroaryl. In some embodiments,monocyclic heteroaryl is a 5- membered or 6-membered heteroaryl. In someembodiments, a bicyclic heteroaryl is a C₆-C₉ heteroaryl. In someembodiments, a heteroaryl group is partially reduced to form aheterocycloalkyl group defined herein. In some embodiments, a heteroarylgroup is fully reduced to form a heterocycloalkyl group defined herein.

As used herein, the “N/P ratio” is the molar ratio of ionizable (e.g.,in the physiological pH range) nitrogen atoms in a lipid (or lipids) tophosphate groups in a nucleic acid molecular entity (or nucleic acidmolecular entities), e.g., in a nanoparticle composition comprising alipid component and an RNA. Ionizable nitrogen atoms can include, forexample, nitrogen atoms that can be protonated at about pH 1, about pH2, about pH 3, about pH 4, about pH5, about pH 6, about pH 7, about pH7.5, or about pH 8 or higher. The physiological pH range can include,for example, the pH range of different cellular compartments (such asorgans, tissues, and cells) and bodily fluids (such as blood, CSF,gastric juice, milk, bile, saliva, tears, and urine). In certainspecific embodiments, the physiological pH range refers to the pH rangeof blood in a mammal, for example, from about 7.35 to about 7.45.Similarly, for phosphate charge neutralizers that have one or moreionizable nitrogen atoms, the N/P ratio can refer to a molar ratio ofionizable nitrogen atoms in the phosphate charge neutralizer to thephosphate groups in a nucleic acid. In some embodiments, ionizablenitrogen atoms refer to those nitrogen atoms that are ionizable within apH range between 5 and 14.

It is further contemplated that the LNP formulations that encapsulatethe gRNAs and mRNAs drug substances described herein may be modified toinclude GalNac lipid formulations such as those disclosed in co-ownedU.S. patent application Ser. No. 17/192,709 filed on Mar. 4, 2021 with aparallel PCT application being filed on same date having InternationalApplication Number PCT/US21/20955. Employment of such GalNac lipidformulations are understood to be capable of enhancing LNP uptake inLDL-R deficient cells, such as those associated with heterozygous andhomozygous familial hypercholesterolemia patient populations.

For the payload that does not contain a phosphate group, the N/P ratiocan refer to a molar ratio of ionizable nitrogen atoms in a lipid to thetotal negative charge in the payload. For example, the N/P ratio of anLNP composition can refer to a molar ratio of the total ionizablenitrogen atoms in the LNP composition to the total negative charge inthe payload that is present in the composition.

As used herein, amino lipids can contain at least one primary, secondaryor tertiary amine moiety that is protonatable (or ionizable) between pHrange 4 and 14. In some embodiments, the amine moiety/moieties functionas the hydrophilic headgroup of the amino lipids. When most of the aminemoiety(ies) of an amino lipid (or amino lipids) in a nucleic acid- lipidnanoparticle formulation is protonated at physiological pH, then thenanoparticles can be termed as cationic lipid nanoparticle (cLNP). Whenmost of the amine moiety(ies) of an amino lipid (or amino lipids) in anucleic acid-lipid nanoparticle formulation is not protonated atphysiological pH but can be protonated at acidic pH, endosomal pH forexample, can be termed as ionizable lipid nanoparticle (iLNP). The aminolipids that constitute cLNPs can be generally called cationic aminolipids (cLipids). The amino lipids that constitute iLNPs can be calledionizable amino lipids (iLipids). The amino lipid can be an iLipid or acLipid at physiological pH.

Kits

One aspect of the disclosure relates to kits including the compositionscomprising a single guide RNA as provided herein, the base editor systemand complex as provided herein, the composition as provided herein, orthe lipid nanoparticle as provided herein for treating or preventing acondition. The kits can further include one or more additionaltherapeutic regimens or agents for treating or preventing a condition.

Also disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods described herein. Such kitsinclude a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. In one embodiment, thecontainers are formed from a variety of materials such as glass orplastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include the composition of the disclosure,and optionally in addition with therapeutic regimens or agents disclosedherein. Such kits optionally include an identifying description or labelor instructions relating to its use in the methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In embodiments, a label is on or associated with the container. In oneembodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself, a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

EXAMPLES Introduction

The present disclosure will be described in greater detail by way ofspecific examples. These examples are provided for illustrative purposesonly and not to limit the scope of the claims provided herein. Those ofskill in the art will readily recognize a variety of non-criticalparameters that can be changed or modified to yield alternativeembodiments according to the present disclosure.

Gene-editing technologies, including CRISPR-Cas9 nucleases and CRISPRbase editors, have the potential to precisely and permanently modifydisease-causing genes in human patients. The demonstration of durableediting in target organs of non-human primates (NHPs) is an importantand necessary step prior to in vivo administration of gene editors topatients in clinical trials. Herein provided is the first demonstrationthat CRISPR base editors delivered in vivo using lipid nanoparticles(LNP) can efficiently modify disease-related genes in living NHPs. Inaddition to providing proof-of-concept for a once-and-done approach toreduce cholesterol and treat coronary heart disease, the leading causeof death worldwide, these results demonstrate how CRISPR base editorscan be productively applied to a variety of therapeutic target genes inthe liver and potentially other organs.

In vivo gene editing is an emerging new therapeutic approach to make DNAmodifications in a patient's own body in organs such as the liver.Gene-editing methods include CRISPR-Cas9 and -Cas12 nucleases, CRISPRcytosine base editors, CRISPR adenine base editors, and CRISPR primeeditors. The human PCSK9 and ANGPTL3 gene are particularly attractivepotential target(s) for in vivo gene editing. In principle, editing ofPCSK9 could produce durable reductions in blood LDL-C levels and therebydramatically lower one's cumulative exposure to LDL-C in contrast to allexisting therapies (e.g., statins, ezetimibe, or PCSK9 inhibitors),which must be taken daily or every few weeks to months and can sufferfrom lack of patient adherence.

In vivo delivery of a gene editor that can introduce a PCSK9 or ANGPTL3loss-of-function mutation in the liver has the potential to offer a newkind of once-and-done therapy that offers lifelong treatment of coronaryheart disease. CRISPR base editors make an attractive gene-editingmodality for this purpose because they function efficiently forintroducing precise targeted alterations and, in contrast to CRISPR-Cas9and other gene-editing nucleases, minimize any deleterious consequencesof introducing double-strand DNA breaks.

CRISPR adenine base editors can induce targeted A→G edits in DNA (T→C onthe opposing strand) and can be used to inactivate genes by disruptingstart codons, splice donors (canonical GT sequence on the sense strand)or splice acceptors (canonical AG sequence on the sense strand) atexon-intron or intron-exon boundaries, or introducing missensemutations. Adenine 8.8-m (hereafter referred to as ABE8.8) uses its coreStreptococcus pyogenes nickase Cas9 (nSpCas9) protein with a guide RNA(gRNA) to engage a double-strand protospacer DNA sequence, flanked by anNGG protospacer-adjacent motif (PAM) sequence on its 3′ end. Theprotospacer sequence is specified via hybridization of the first 20bases of the gRNA with a complementary sequence on the “target” DNAstrand, leaving part of the other (“non-target”) strand in exposedsingle-strand form structure called the R-loop. The ABE base editor usesan evolved deoxyadenosine deaminase domain—fused to nSpCas9—tochemically modify an adenosine nucleoside, contained in thesingle-stranded DNA portion of the R-loop, into inosine and nicks thetarget DNA strand within the DNA:RNA heteroduplex of the R-loop. Thisnick biases DNA repair machinery to use the freshly deaminated strand asa template, enabling highly efficient transition mutation at thetargeted site. The activity window of ABE8.8 typically ranges frompositions 3 to 9 in the protospacer DNA sequence specified by the gRNA,12 to 18 base pairs 5′ of the NGG PAM (positions 21 to 23), with peakediting observed at position 6 of the protospacer. Although themechanism of action of ABE8.8 does not involve double-strand DNA breaks,indel mutagenesis can occur low frequency.

The advantages of base editing compared to CRISPR-Cas9 genome editingfor gene inactivation or other types of gene alteration is noted.Standard CRISPR-Cas9 editing carries higher risks of unwanted on-targeteffects and off-target effects emanating from double-strand breaks(large deletions, integration of vector DNA sequences, chromosomalrearrangements, induction of p53 activity, etc.). Even the intendedon-target effects-small indel mutations—have an element ofunpredictability, as they can result in frameshift mutations that addvaried strings of anomalous amino acids at the end of the truncatedprotein product, or in-frame mutations that add or remove amino acidsfrom the protein without knocking out its function. In contrast, baseediting offers a means to efficiently make precise, stereotyped changesin the genome, allowing for more reproducible alteration of genefunction. Base editing mitigates unintended on-target effects by virtueof not requiring double-strand breaks but instead acting throughenzymatic modification of DNA bases via the deaminase domain.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents.

Protospacer Selection and Editing Example 1. gRNA Design for TargetingPCSK9 and ANGPTL3

All protospacers shown in Table 1 and Table 24 were chosen by twocriteria. First, they matched (or very closely matched) sequences in thehuman, cynomolgus monkey, and/or mouse orthologs of the genes. Second,they had favorable predicted off-target profiles, as judged by the MITSpecificity Score (calculated by http://crispor.tefor.net/; minimumscore of 50).

Where the goal is to disrupt a gene in vivo for a therapeutic purpose,cytosine base editors have the advantage of being able to directlyintroduce stop codons into the coding sequence of the gene (nonsensemutations) by altering specific codons for glutamine (CAG→TAG, CAA→TAA),arginine (CGA→TGA), and tryptophan (TGG→TAG/TAA/TGA, with editing ofcytosines on the antisense strand). In contrast, adenine base editorscannot directly introduce stop codons, as there are no A→G changes thatresult in nonsense mutations. Nonetheless, the more favorable off-targetprofile of adenine base editors, particularly with respect togRNA-independent off-target DNA base editing, recommend the use ofadenine base editors over cytosine base editors for therapeuticpurposes. Similar approaches were followed to identify and selectmouse/rodent specific protospacers listed in Tables 1 and 24.

One strategy by which adenine base editors might be used to disrupt genefunction is to edit the start codon, such as from ATG→GTG or ATG→ACG.Therefore, the resultant translation into protein will not initiate atthe canonical ATG site. A second strategy by which adenine base editorsmight be used to disrupt gene function is to edit splice sites, whethersplice donors at the 5′ ends of introns or splice acceptors at the 3′ends of introns. Splice site disruption can result in the inclusion ofintronic sequences in messenger RNA (mRNA) potentially introducingnonsense, frameshift, or in-frame indel mutations that result inpremature stop codons or in insertion/deletion of amino acids thatdisrupt protein activity—or in the exclusion of exonic sequences, whichcan also introduce nonsense, frameshift, or in-frame indel mutations.Canonical splice donors comprise the DNA sequence GT on the sensestrand, whereas canonical splice acceptors comprise the DNA sequence AG.Alteration of the sequence disrupts normal splicing. Splice donors canbe disrupted by adenine base editing of the complementary base in thesecond position in the antisense strand (GT→GC), and splice acceptorscan be disrupted by adenine base editing of the first position in thesense strand (AG→GG). A third strategy by which adenine base editorsmight be used to disrupt gene function is to introduce a missensemutation(s) into the coding region of the gene that results inproduction of a less functional, or non-functional protein.

All gRNA spacer sequences that would permit ABE8.8 (and other ABEvariants containing Streptococcus pyogenes Cas9, such as ABE7.10, oranother Cas protein that can use the NGG PAMN) to disrupt splice siteswere identified, whether donors or acceptors, via A→G editing within itsediting window (roughly positions 3 to 9 in the 20-nt protospacer regionof DNA) were identified. Guide RNAs matching each of the protospacersequences and otherwise conforming to the standard 100-nt Streptococcuspyogenes CRISPR gRNA sequence were synthesized, with each gRNA moleculehaving a modest degree of chemical modifications (e.g., in Table 1).

TABLE 1 Guide RNAs (SgRNA/gRNA) for Cas9 nuclease and ABE editors Proto-SEQ SEQ Editor Gene gRNA spacer ID ID associated Target ID (5′-3′) NOSequence (5′-3′) NO data hcPCSK9 GA001 GGTGCTAG 66GsGsUsGCUAGCCUUGCGUUCCG 253 SpCas9 CCTTGCGT GUUUUAGAgcuagaaauagcAAGUUTCCG AAAAUAAGGCUAGUCCGUUAU CAacuugaaaaaguggcaccgagucggugcu sususuhcPCSK9 GA002 GGTGCTAG 66 gsgsusGCUAGCCUUGCGUUCCGG 253 SpCas9 CCTTGCGTUUUUAGAgcuagaaauagcAAGUUA TCCG AAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusu susu hcPCSK9 GA003 GGTGCTAG 66gsgsusGCUAGCCUUGCGUUCCGG 254 SpCas9 CCTTGCGT UUUUsAGAgcuagaaauagcAAGUUsTCCG AAAAUsAAGGCUsAGUCCGUUs AUCsAacuugaaaaaguggcaccgagucgg ugcusususuhcPCSK9 GA004 GGTGCTAG 66 gsgsusGCUAGCCUUGCGUUCCGg 255 SpCas9 CCTTGCGTUUUUAGagcuagaaauagcaaGUUaAa TCCG AuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcuuuuu hcPCSK9 GA005 GGTGCTAG 66GsGsUsGCUAGCCUUGCGUUCCG 255 SpCas9 CCTTGCGT gUUUUAGagcuagaaauagcaaGUUaATCCG aAuAaggcuaGUccGUUAucAAcuuga aaaagugGcaccgagucggugcuuuuu hcPCSK9GA006 GGTGCTAG 66 GsGsUsGCUAGCCUUGCGUUCCG 253 SpCas9 CCTTGCGTgUUUUAGagcuagaaauagcaaGUUaA TCCG aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu hcPCSK9 GA007 GGTGCTAG 66gsgsusGCUAGCCUUGCGUUCCGg 253 SpCas9 CCTTGCGT UUUUAGagcuagaaauagcaaGUUaAaTCCG AuAaggcuaGUccGUUAucAAcuugaa aaagugGcaccgagucggugcusususu hcPCSK9GA007/ GGTGCTAG 66 gsgsusGCUAGCCUUGCGUUCCGg 253 SpCas9 GA252 CCTTGCGTUUUUAGagcuagaaauagcaaGUUaAa TCCG AuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu hcPCSK9 GA008 GGTGCTAG 66gsgsusGCUAGCCUUGCGUUCCGg 256 SpCas9 CCTTGCGT UUUUAGagcuagaaauagcaaGUUsaATCCG aAuAaggcuaGUccGUUsAucAAcuug aaaaagugGcaccgagucggugcusususu hcPCSK9GA009 GGTGCTAG 66 gsgsusGCUAGCCUUGCGUUCCGG 253 SpCas9 CCTTGCGTUUUUAGAGCUAGAAAUAGCAA TCCG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu mcPCSK9 GA052 CAGGTTCC 81 csasgsGUUCCAUGGGAUGCUCUG55 SpCas9 ATGGGATG UUUUAGAGCUAGAAAUAGCAA CTCT GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu mcPCSK9 GA053 CAGGTTCC 81csasgsGUUCCAUGGGAUGCUCUG 55 SpCas9 ATGGGATG UUUUAGAgcuagaaauagcAAGUUACTCT AAAUAAGGCUAGUCCGUUAUC Aacuugaaaaaguggcaccgagucggugcusu susu mcPCSK9GA054 CAGGTTCC 81 csasgsGUUCCAUGGGAUGCUCUg 55 SpCas9 ATGGGATGUUUUAGagcuaGaaauagcaaGUUaAa CTCT AuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu mcPCSK9 GA055 CAGGTTCC 81csasgsGUUCCAUGGGAUGCUCUg 55 SpCas9 ATGGGATG UUUUAGagcuagaaauagcaaGUUaAaCTCT AuAaggcuaGUccGUUAucAAcuugaa aaagugGcaccgagucggugcusususu hcPCSK9GA066/ CCCGCACC 13 cscscsGCACCUUGGCGCAGCGGG 9 ABE/ GA095 TTGGCGCAUUUUAGAgcuagaaauagcAAGUUA SpCas9 GCGG AAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusu susu cANGPTL3 GA067/ AAGATACC 14asasgsAUACCUGAAUAACUCUCG 257 ABE/ GA101 TGAATAACUUUUAGAgcuagaaauagcAAGUUA SpCas9 TCTC AAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusu susu hcPCSK9 GA072 GGTGGCTC 82gsgsusGGCUCACCAGCUCCAGCG 258 ABE ACCAGCTC UUUUAGAGCUAGAAAUAGCAA CAGCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA073 GCTTACCT 67 gscsusUACCUGUCUGUGGAAGC 259 ABE GTCTGTGGGUUUUAGAGCUAGAAAUAGCA AAGC AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA074 TGCTTACC 68 usgscsUUACCUGUCUGUGGAAG260 ABE TGTCTGTG GUUUUAGAGCUAGAAAUAGCA GAAG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hPCSK9 GA075 TTGGAAAG 83ususgsGAAAGACGGAGGCAGCC 261 ABE ACGGAGG GUUUUAGAGCUAGAAAUAGCA CAGCCAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hPCSK9GA076 GAAAGAC 84 gsasasAGACGGAGGCAGCCUGGG 262 ABE GGAGGCAUUUUAGAGCUAGAAAUAGCAA GCCTGG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA077 TCCCAGGC 85 uscscsCAGGCCUGGAGUUUAUU263 ABE CTGGAGTT GUUUUAGAGCUAGAAAUAGCA TATT AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hPCSK9 GA078 AGCACCTA 86asgscsACCUACCUCGGGAGCTGG 264 ABE CCTCGGGA UUUUAGAGCUAGAAAUAGCAA GCTGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA079 CTTTCCAG 87 csususUCCAGGUCAUCACAGUUG 265 ABE GTCATCACUUUUAGAGCUAGAAAUAGCAA AGTT GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA080 CCTTTCCA 88 cscsusUUCCAGGUCAUCACAGUG266 ABE GGTCATCA UUUUAGAGCUAGAAAUAGCAA CAGT GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA081 TTTCCAGG 89usususCCAGGUCAUCACAGUUG 267 ABE TCATCACA GUUUUAGAGCUAGAAAUAGCA GTTGAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hPCSK9GA082 CTTACCTG 90 csususACCUGCCCCAUGGGUGCG 268 ABE CCCCATGGUUUUAGAGCUAGAAAUAGCAA GTGC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hPCSK9 GA083 TAAGGCCC 91 usasasGGCCCAAGGGGGCAAGCG269 ABE AAGGGGG UUUUAGAGCUAGAAAUAGCAA CAAGC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hPCSK9 GA084 CCTCTTCA 92cscsusCUUCACCUGCUCCUGAGG 270 ABE CCTGCTCC UUUUAGAGCUAGAAAUAGCAA TGAGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hPCSK9GA085 GCCTCTTC 93 gscscsUCUUCACCUGCUCCUGAG 271 ABE ACCTGCTCUUUUAGAGCUAGAAAUAGCAA CTGA GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hPCSK9 GA086 TTCACCTG 94 ususcsACCUGCUCCUGAGGGGCG272 ABE CTCCTGAG UUUUAGAGCUAGAAAUAGCAA GGGC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA087 TCACCTGC 95uscsasCCUGCUCCUGAGGGGCCG 273 ABE TCCTGAGG UUUUAGAGCUAGAAAUAGCAA GGCCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA088 CCCAGGCT 96 cscscsAGGCUGCAGCUCCCACUG 274 ABE GCAGCTCCUUUUAGAGCUAGAAAUAGCAA CACT GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hPCSK9 GA089 CCCCAGGC 97 cscscsCAGGCUGCAGCUCCCACG275 ABE TGCAGCTC UUUUAGAGCUAGAAAUAGCAA CCAC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hPCSK9 GA090 GCAGGTG 98gscsasGGUGACCGUGGCCUGCGG 276 ABE ACCGTGGC UUUUAGAGCUAGAAAUAGCAA CTGCGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hANGPTL3GA091 AAGATACC 15 asasgsAUACCUGAAUAACCCUCG 59 ABE TGAATAACUUUUAGAGCUAGAAAUAGCAA CCTC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcANGPTL3 GA092 CTCCTTTA 99 csuscsCUUUAGGAGGCUGGUGG277 ABE GGAGGCT GUUUUAGAGCUAGAAAUAGCA GGTGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hANGPTL3 GA093 TTTTCAGG 100usususUCAGGAGAAUUUUGGUU 278 ABE AGAATTTT GUUUUAGAGCUAGAAAUAGCA GGTTAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA094/ CTTTTCAG 101 csususUUCAGGAGAAUUUUGGU 279 ABE GA153GAGAATTT GUUUUAGAGCUAGAAAUAGCA TGGT AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA095/ CCCGCACC 13cscscsGCACCUUGGCGCAGCGGG 9 ABE GA066 TTGGCGCA UUUUAGAgcuagaaauagcAAGUUAGCGG AAAUAAGGCUAGUCCGUUAUC Aacuugaaaaaguggcaccgagucggugcusu susu hcPCSK9GA096 CCCGCACC 13 cscscsGCACCUUGGCGCAGCGGg 9 ABE TTGGCGCAUUUUAGagcuaGaaauagcaaGUUaAa GCGG AuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu hcPCSK9 GA097 CCCGCACC 13cscscsGCACCUUGGCGCAGCGGg 9 ABE TTGGCGCA UUUUAGagcuagaaauagcaaGUUaAa GCGGAuAaggcuaGUccGUUAucAAcuugaa aaagugGcaccgagucggugcusususu hANGPTL3 GA098AAGATACC 15 asasgsAUACCUGAAUAACCCUCG 59 ABE TGAATAACUUUUAGAgcuagaaauagcAAGUUA CCTC AAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusu susu hANGPTL3 GA099 AAGATACC 15asasgsAUACCUGAAUAACCCUCg 59 ABE TGAATAAC UUUUAGagcuaGaaauagcaaGUUaAaCCTC AuAaggCUaGUCcGUUAucAAcuuG aaaaaguGgcaccgAgUCggugcusususu hANGPTL3GA100 AAGATACC 15 asasgsAUACCUGAAUAACCCUCg 59 ABE TGAATAACUUUUAGagcuagaaauagcaaGUUaAa CCTC AuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu cANGPTL3 GA101/ AAGATACC 14asasgsAUACCUGAAUAACUCUCG 257 ABE GA067 TGAATAACUUUUAGAgcuagaaauagcAAGUUA TCTC AAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusu susu cANGPTL3 GA102 AAGATACC 14asasgsAUACCUGAAUAACUCUCg 257 ABE TGAATAAC UUUUAGagcuaGaaauagcaaGUUaAaTCTC AuAaggCUaGUCcGUUAucAAcuuG aaaaaguGgcaccgAgUCggugcusususu cANGPTL3GA103 AAGATACC 14 asasgsAUACCUGAAUAACUCUCg 257 ABE TGAATAACUUUUAGagcuagaaauagcaaGUUaAa TCTC AuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu hANGPTL3 GA104 TAATTTGG 102usasasUUUGGCCCUUCGUCUUAG 280 ABE CCCTTCGT UUUUAGAGCUAGAAAUAGCAA CTTAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hANGPTL3GA105 AGACTTTG 103 asgsasCUUUGUCCAUAAGACGAG 281 ABE TCCATAAGUUUUAGAGCUAGAAAUAGCAA ACGA GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hANGPTL3 GA106 GACTTTGT 104 gsascsUUUGUCCAUAAGACGAA282 ABE CCATAAGA GUUUUAGAGCUAGAAAUAGCA CGAA AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA107 AGCCAATG 105asgscsCAAUGGCCUCCUUCAGUG 283 SpCas9 GCCTCCTT UUUUAGAGCUAGAAAUAGCAA CAGTGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususuhcANGPTL3 GA108 TCCCAACT 106 uscscsCAACUGAAGGAGGCCAUG 284 SpCas9 GAAGGAGUUUUAGAGCUAGAAAUAGCAA GCCAT GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcANGPTL3 GA109 GGCCTCCT 107gsgscsCUCCUUCAGUUGGGACAG 285 SpCas9 TCAGTTGG UUUUAGAGCUAGAAAUAGCAA GACAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususuhcANGPTL3 GA110 GACCATGT 108 gsascsCAUGUCCCAACUGAAGGG 286 SpCas9CCCAACTG UUUUAGAGCUAGAAAUAGCAA AAGG GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcANGPTL3 GA111 GCCAATGG 109gscscsAAUGGCCUCCUUCAGUUG 287 SpCas9 CCTCCTTC UUUUAGAGCUAGAAAUAGCAA AGTTGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hANGPTL3GA112 ATTGTCTT 110 asususGUCUUGAUCAAUUCUGG 288 ABE/ GATCAATTGUUUUAGAGCUAGAAAUAGCA SpCas9 CTGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA113 ATTCTGGA 111asususCUGGAGGAAAUAACUAG 289 ABE/ GGAAATA GUUUUAGAGCUAGAAAUAGCA SpCas9ACTAG AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA114 TCTGGGTG 112 uscsusGGGUGUUCUGGAGUUUC 290 SpCas9 TTCTGGAGGUUUUAGAGCUAGAAAUAGCA TTTC AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcANGPTL3 GA115 AACATAGC 113asascsAUAGCAAAUCUUGAUUU 291 ABE/ AAATCTTG GUUUUAGAGCUAGAAAUAGCA SpCas9ATTT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA116 GTAGAATT 114 gsusasGAAUUUUUUCUUCUAGG 292 ABE/ TTTTCTTCGUUUUAGAGCUAGAAAUAGCA SpCas9 TAGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA117 ACTACAAG 115ascsusACAAGUCAAAAAUGAAG 293 ABE/ TCAAAAAT GUUUUAGAGCUAGAAAUAGCA SpCas9GAAG AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA118 TATATTGG 116 usasusAUUGGUCUUCCACGGUCG 294 ABE/ TCTTCCACUUUUAGAGCUAGAAAUAGCAA SpCas9 GGTC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hANGPTL3 GA119 CAAAGAC 117csasasAGACCUUCUCCAGACCGG 295 ABE/ CTTCTCCA UUUUAGAGCUAGAAAUAGCAA SpCas9GACCG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususuhANGPTL3 GA120 GGTCTTCC 118 gsgsusCUUCCACGGUCUGGAGA 296 ABE/ ACGGTCTGGUUUUAGAGCUAGAAAUAGCA SpCas9 GAGA AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hANGPTL3 GA121 TTGTTTAT 119ususgsUUUAUAUUGGUCUUCCA 297 ABE/ ATTGGTCT GUUUUAGAGCUAGAAAUAGCA SpCas9TCCA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA122 CTTTTATT 120 csususUUAUUUGACUAUGCUGU 298 ABE/ TGACTATGGUUUUAGAGCUAGAAAUAGCA SpCas9 CTGT AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hANGPTL3 GA123 AAAGTCTG 121asasasGUCUGGAUAUAGAGAGU 299 ABE/ GATATAGA GUUUUAGAGCUAGAAAUAGCA SpCas9GAGT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA124 GTTGGTTT 122 gsususGGUUUAAUUGUUUAUAU 300 ABE/ AATTGTTTGUUUUAGAGCUAGAAAUAGCA SpCas9 ATAT AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA125 TGATGGTA 123usgsasUGGUAAGACACUUUGGU 301 ABE/ AGACACTT GUUUUAGAGCUAGAAAUAGCA SpCas9TGGT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA126 GGAGTAGT 124 gsgsasGUAGUUCUUGGUGCUCU 302 ABE/ TCTTGGTGGUUUUAGAGCUAGAAAUAGCA SpCas9 CTCT AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA127 AACATGAT 125asascsAUGAUGGUAAGACACUU 303 ABE/ GGTAAGA GUUUUAGAGCUAGAAAUAGCA SpCas9CACTT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA128 TGAAGAA 126 usgsasAGAAAGGGAGUAGUUCU 304 ABE/ AGGGAGTGUUUUAGAGCUAGAAAUAGCA SpCas9 AGTTCT AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA129 AGTTCTTG 127asgsusUCUUGGUGCUCUUGGCU 305 ABE/ GTGCTCTT GUUUUAGAGCUAGAAAUAGCA SpCas9GGCT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA130 ATGATGGT 128 asusgsAUGGUAAGACACUUUGG 306 ABE/ AAGACACTGUUUUAGAGCUAGAAAUAGCA SpCas9 TTGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA131 GAAGATA 129gsasasGAUAGAGAAAUUUCUGU 307 ABE/ GAGAAATT GUUUUAGAGCUAGAAAUAGCA SpCas9TCTGT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA132 GGAAGAT 130 gsgsasAGAUAGAGAAAUUUCUG 308 ABE/ AGAGAAAGUUUUAGAGCUAGAAAUAGCA SpCas9 TTTCTG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hANGPTL3 GA133 TATTTCAT 131usasusUUCAUUCAACUGAAGAA 309 ABE/ TCAACTGA GUUUUAGAGCUAGAAAUAGCA SpCas9AGAA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA134 ATTTCATT 132 asususUCAUUCAACUGAAGAAA 310 ABE/ CAACTGAAGUUUUAGAGCUAGAAAUAGCA SpCas9 GAAA AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hANGPTL3 GA135 GTCTACTG 133gsuscsUACUGUGAUGUUAUAUC 311 ABE/ TGATGTTA GUUUUAGAGCUAGAAAUAGCA SpCas9TATC AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA136 TAAATGGT 134 usasasAUGGUGGUACAUUCAGC 312 ABE/ GGTACATTGUUUUAGAGCUAGAAAUAGCA SpCas9 CAGC AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA137 TATCAGGT 135usasusCAGGUAAAACCUGUCUA 313 ABE/ AAAACCTG GUUUUAGAGCUAGAAAUAGCA SpCas9TCTA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA138 TGTACCAC 136 usgsusACCACCAUUUAUAACAG 314 ABE/ CATTTATAGUUUUAGAGCUAGAAAUAGCA SpCas9 ACAG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hANGPTL3 GA139 TTCACCTC 137ususcsACCUCUGUUAUAAAUGG 315 ABE/ TGTTATAA GUUUUAGAGCUAGAAAUAGCA SpCas9ATGG AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA140 AACAGAG 138 asascsAGAGGUGAACAUACAAG 316 ABE/ GTGAACATGUUUUAGAGCUAGAAAUAGCA SpCas9 ACAAG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA141 TTGAGAGT 139ususgsAGAGUUGCUGGGUCUGA 317 ABE/ TGCTGGGT GUUUUAGAGCUAGAAAUAGCA SpCas9CTGA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA142 TGAAAAA 140 usgsasAAAACUUGAGAGUUGCU 318 SpCas9 CTTGAGAGGUUUUAGAGCUAGAAAUAGCA TTGCT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcANGPTL3 GA143 TTAATTCA 141ususasAUUCAACAUCGAAUAGA 319 ABE/ ACATCGAA GUUUUAGAGCUAGAAAUAGCA SpCas9TAGA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA144 TTTGGGAG 142 usususGGGAGGCUUGAUGGUAA 320 ABE/ GCTTGATGGUUUUAGAGCUAGAAAUAGCA SpCas9 GTAA AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA145 CATTATAT 143csasusUAUAUUCAGGUAGUCCA 321 ABE/ TCAGGTAG GUUUUAGAGCUAGAAAUAGCA SpCas9TCCA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA146 TTGGGAGG 144 ususgsGGAGGCUUGAUGGUAAG 322 ABE/ CTTGATGGGUUUUAGAGCUAGAAAUAGCA SpCas9 TAAG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hANGPTL3 GA147 TTTTGGGA 145usususUGGGAGGCUUGAUGGUA 323 ABE/ GGCTTGAT GUUUUAGAGCUAGAAAUAGCA SpCas9GGTA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA148 ACAAAACT 146 ascsasAAACUUCAAUGAAACGUG 324 ABE/ TCAATGAAUUUUAGAGCUAGAAAUAGCAA SpCas9 ACGT GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hANGPTL3 GA149 TATGGTTT 147usasusGGUUUUGGGAGGCUUGA 325 ABE/ TGGGAGG GUUUUAGAGCUAGAAAUAGCA SpCas9CTTGA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA150 ACTACAAA 148 ascsusACAAAUAUGGUUUUGGG 326 ABE/ TATGGTTTGUUUUAGAGCUAGAAAUAGCA SpCas9 TGGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcANGPTL3 GA151 GAGAACT 149gsasgsAACUACAAAUAUGGUUU 327 ABE/ ACAAATAT GUUUUAGAGCUAGAAAUAGCA SpCas9GGTTT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhANGPTL3 GA152 AGGACACT 150 asgsgsACACUUCAACUGUCCAGG 328 ABE/ TCAACTGTUUUUAGAGCUAGAAAUAGCAA SpCas9 CCAG GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hANGPTL3 GA094/ CTTTTCAG 101csususUUCAGGAGAAUUUUGGU 279 ABE/ GA153 GAGAATTT GUUUUAGAGCUAGAAAUAGCASpCas9 TGGT AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA156 GGTGCTAG 66 gsgsusGCUAGCCUUGCGUUCCGG253 SpCas9 CCTTGCGT UUUUAGAGCUAGAAAUAGCAA TCCG GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA157 GCCGTCCT 151gscscsGUCCUCCUCGGAACGCAG 329 SpCas9 CCTCGGAA UUUUAGAGCUAGAAAUAGCAA CGCAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA158 GCTAGCCT 152 gscsusAGCCUUGCGUUCCGAGGG 330 SpCas9 TGCGTTCCUUUUAGAGCUAGAAAUAGCAA GAGG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA159 GCGTTCCG 153 gscsgsUUCCGAGGAGGACGGCCG331 SpCas9 AGGAGGA UUUUAGAGCUAGAAAUAGCAA CGGCC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA160 GCCTTGCG 154gscscsUUGCGUUCCGAGGAGGA 332 SpCas9 TTCCGAGG GUUUUAGAGCUAGAAAUAGCA AGGAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9GA161 GGACGAG 155 gsgsasCGAGGACGGCGACUACGG 333 SpCas9 GACGGCGUUUUAGAGCUAGAAAUAGCAA ACTACG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA162 GGACGGC 156 gsgsasCGGCGACUACGAGGAGCG334 SpCas9 GACTACGA UUUUAGAGCUAGAAAUAGCAA GGAGC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA163 CGTCCTCG 157csgsusCCUCGUCCUCCUGCGCAG 335 SpCas9 TCCTCCTG UUUUAGAGCUAGAAAUAGCAA CGCAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA164 GTCCTCGT 158 gsuscsCUCGUCCUCCUGCGCACG 336 SpCas9 CCTCCTGCUUUUAGAGCUAGAAAUAGCAA GCAC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA165 CCGTCAGC 159 cscsgsUCAGCUCCAGGCGGUCCG337 SpCas9 TCCAGGCG UUUUAGAGCUAGAAAUAGCAA GTCC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA166 CGCCCGTG 160csgscsCCGUGCGCAGGAGGACGG 338 SpCas9 CGCAGGA UUUUAGAGCUAGAAAUAGCAA GGACGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA167 TCAGCTCC 161 uscsasGCUCCAGGCGGUCCUGGG 339 SpCas9 AGGCGGTCUUUUAGAGCUAGAAAUAGCAA CTGG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA168 CTTGGCGC 162 csususGGCGCAGCGGUGGAAGG340 SpCas9 AGCGGTG GUUUUAGAGCUAGAAAUAGCA GAAGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA169 CGTGCGCA 163csgsusGCGCAGGAGGACGAGGA 341 SpCas9 GGAGGAC GUUUUAGAGCUAGAAAUAGCA GAGGAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9GA170 CCAGGACC 164 cscsasGGACCGCCUGGAGCUGAG 342 SpCas9 GCCTGGAGUUUUAGAGCUAGAAAUAGCAA CTGA GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA171 TCTTGGTG 165 uscsusUGGUGAGGUAUCCCCGG343 SpCas9 AGGTATCC GUUUUAGAGCUAGAAAUAGCA CCGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA172 CAGTGCGC 166csasgsUGCGCUCUGACUGCGAGG 344 SpCas9 TCTGACTG UUUUAGAGCUAGAAAUAGCAA CGAGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA173 CTTGGTGA 167 csususGGUGAGGUAUCCCCGGCG 345 SpCas9 GGTATCCCUUUUAGAGCUAGAAAUAGCAA CGGC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA174 GGATCTTG 168 gsgsasUCUUGGUGAGGUAUCCC346 SpCas9 GTGAGGTA GUUUUAGAGCUAGAAAUAGCA TCCC AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA175 GAAGATG 169gsasasGAUGAGUGGCGACCUGCG 347 SpCas9 AGTGGCG UUUUAGAGCUAGAAAUAGCAA ACCTGCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA176 AGCACCAC 170 asgscsACCACCACGUAGGUGCCG 348 SpCas9 CACGTAGGUUUUAGAGCUAGAAAUAGCAA TGCC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA177 GGTCGCCA 171 gsgsusCGCCACUCAUCUUCACCG349 SpCas9 CTCATCTT UUUUAGAGCUAGAAAUAGCAA CACC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA178 CTCCTTCA 172csuscsCUUCAGCACCACCACGUG 350 SpCas9 GCACCACC UUUUAGAGCUAGAAAUAGCAA ACGTGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA179 GAGTGGC 173 gsasgsUGGCGACCUGCUGGAGCG 351 SpCas9 GACCTGCTUUUUAGAGCUAGAAAUAGCAA GGAGC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA180 GCGCACTG 174 gscsgsCACUGCCCGCCGCCUGCG352 SpCas9 CCCGCCGC UUUUAGAGCUAGAAAUAGCAA CTGC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA181 GGCTTCCT 175gsgscsUUCCUGGUGAAGAUGAG 353 SpCas9 GGTGAAG GUUUUAGAGCUAGAAAUAGCA ATGAGAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9GA182 CACCTACG 176 csascsCUACGUGGUGGUGCUGAG 354 SpCas9 TGGTGGTGUUUUAGAGCUAGAAAUAGCAA CTGA GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA183 CTACGTGG 177 csusasCGUGGUGGUGCUGAAGG355 SpCas9 TGGTGCTG GUUUUAGAGCUAGAAAUAGCA AAGG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA184 ATGGAAG 178asusgsGAAGACAUGCAGGAUCU 356 SpCas9 ACATGCAG GUUUUAGAGCUAGAAAUAGCA GATCTAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9GA185 AGACATGC 179 asgsasCAUGCAGGAUCUUGGUG 357 SpCas9 AGGATCTTGUUUUAGAGCUAGAAAUAGCA GGTG AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA186 CTCATCTT 180 csuscsAUCUUCACCAGGAAGCCG358 SpCas9 CACCAGG UUUUAGAGCUAGAAAUAGCAA AAGCC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA187 CTCCTCGA 181csuscsCUCGAUGUAGUCGACAUG 359 SpCas9 TGTAGTCG UUUUAGAGCUAGAAAUAGCAA ACATGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA188 TATTCATC 182 usasusUCAUCCGCCCGGUACCGG 360 SpCas9 CGCCCGGTUUUUAGAGCUAGAAAUAGCAA ACCG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA189 TCCTCGAT 183 uscscsUCGAUGUAGUCGACAUG361 SpCas9 GTAGTCGA GUUUUAGAGCUAGAAAUAGCA CATG AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA190 CCTCCTCG 184cscsusCCUCGAUGUAGUCGACAG 362 SpCas9 ATGTAGTC UUUUAGAGCUAGAAAUAGCAA GACAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA191 GCCCCATG 185 gscscsCCAUGUCGACUACAUCGG 363 SpCas9 TCGACTACUUUUAGAGCUAGAAAUAGCAA ATCG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA192 CCATGTCG 186 cscsasUGUCGACUACAUCGAGGG364 SpCas9 ACTACATC UUUUAGAGCUAGAAAUAGCAA GAGG GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA193 GGGGCTG 187gsgsgsGCUGGUAUUCAUCCGCCG 365 SpCas9 GTATTCAT UUUUAGAGCUAGAAAUAGCAA CCGCCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA194 GTCGACAT 188 gsuscsGACAUGGGGCAACUUCA 366 SpCas9 GGGGCAAGUUUUAGAGCUAGAAAUAGCA CTTCA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA195 ACCACCGG 189 ascscsACCGGGAAAUCGAGGGCG367 SpCas9 GAAATCG UUUUAGAGCUAGAAAUAGCAA AGGGC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA196 GAGTGACC 190gsasgsUGACCACCGGGAAAUCGG 368 SpCas9 ACCGGGA UUUUAGAGCUAGAAAUAGCAA AATCGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA197 AGTGACCA 191 asgsusGACCACCGGGAAAUCGAG 369 SpCas9 CCGGGAAUUUUAGAGCUAGAAAUAGCAA ATCGA GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA198 CCACCGGG 192 cscsasCCGGGAAAUCGAGGGCAG370 SpCas9 AAATCGA UUUUAGAGCUAGAAAUAGCAA GGGCA GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA199 GAAGCGG 193gsasasGCGGGUCCCGUCCUCCUG 371 SpCas9 GTCCCGTC UUUUAGAGCUAGAAAUAGCAA CTCCTGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA200 CTAGGAG 194 csusasGGAGAUACACCUCCACCG 372 SpCas9 ATACACCTUUUUAGAGCUAGAAAUAGCAA CCACC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA201 CAGCATAC 195 csasgsCAUACAGAGUGACCACCG373 SpCas9 AGAGTGA UUUUAGAGCUAGAAAUAGCAA CCACC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA202 AAGCGGG 196asasgsCGGGUCCCGUCCUCCUCG 374 SpCas9 TCCCGTCC UUUUAGAGCUAGAAAUAGCAA TCCTCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA203 TGACCCTG 197 usgsasCCCUGCCCUCGAUUUCCG 375 SpCas9 CCCTCGATUUUUAGAGCUAGAAAUAGCAA TTCC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA204 CACTCTGT 198 csascsUCUGUAUGCUGGUGUCUG376 SpCas9 ATGCTGGT UUUUAGAGCUAGAAAUAGCAA GTCT GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA205 CCCTGCCC 199cscscsUGCCCUCGAUUUCCCGGG 377 SpCas9 TCGATTTC UUUUAGAGCUAGAAAUAGCAA CCGGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA206 CCGGTGGT 200 cscsgsGUGGUCACUCUGUAUGCG 378 SpCas9 CACTCTGTUUUUAGAGCUAGAAAUAGCAA ATGC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA207 GGAAATC 201 gsgsasAAUCGAGGGCAGGGUCA379 SpCas9 GAGGGCA GUUUUAGAGCUAGAAAUAGCA GGGTCA AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA208 CCAGCATA 202cscsasGCAUACAGAGUGACCACG 380 SpCas9 CAGAGTG UUUUAGAGCUAGAAAUAGCAA ACCACGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA209 CTTGGCAG 203 csususGGCAGUUGAGCACGCGCG 381 SpCas9 TTGAGCACUUUUAGAGCUAGAAAUAGCAA GCGC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA210 CTGCGCGT 204 csusgsCGCGUGCUCAACUGCCAG382 SpCas9 GCTCAACT UUUUAGAGCUAGAAAUAGCAA GCCA GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA211 TGCGCGTG 205usgscsGCGUGCUCAACUGCCAAG 383 SpCas9 CTCAACTG UUUUAGAGCUAGAAAUAGCAA CCAAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA212 CGGGATGC 206 csgsgsGAUGCCGGCGUGGCCAAG 384 SpCas9 CGGCGTGGUUUUAGAGCUAGAAAUAGCAA CCAA GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA213 CGTGCTCA 207 csgsusGCUCAACUGCCAAGGGAG385 SpCas9 ACTGCCAA UUUUAGAGCUAGAAAUAGCAA GGGA GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA214 CCTTGGCC 208cscsusUGGCCACGCCGGCAUCCG 386 SpCas9 ACGCCGGC UUUUAGAGCUAGAAAUAGCAA ATCCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA215 CAGCGGCC 209 csasgsCGGCCGGGAUGCCGGCGG 387 SpCas9 GGGATGCCUUUUAGAGCUAGAAAUAGCAA GGCG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA216 CCGGGATG 210 cscsgsGGAUGCCGGCGUGGCCAG388 SpCas9 CCGGCGTG UUUUAGAGCUAGAAAUAGCAA GCCA GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA217 GTGGTCAG 211gsusgsGUCAGCGGCCGGGAUGC 389 SpCas9 CGGCCGG GUUUUAGAGCUAGAAAUAGCA GATGCAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9GA218 CGCTGACC 212 csgscsUGACCACCCCUGCCAGGG 390 SpCas9 ACCCCTGCUUUUAGAGCUAGAAAUAGCAA CAGG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA219 GGCAGGG 213 gsgscsAGGGGUGGUCAGCGGCC391 SpCas9 GTGGTCAG GUUUUAGAGCUAGAAAUAGCA CGGCC AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA220 GTGCTCAA 214gsusgsCUCAACUGCCAAGGGAA 392 SpCas9 CTGCCAAG GUUUUAGAGCUAGAAAUAGCA GGAAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hPCSK9GA221 TCATGGCA 215 uscsasUGGCACCCACCUGGCAGG 393 SpCas9 CCCACCTGUUUUAGAGCUAGAAAUAGCAA GCAG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA222 TGGCAGG 216 usgsgsCAGGGGUGGUCAGCGGC394 SpCas9 GGTGGTCA GUUUUAGAGCUAGAAAUAGCA GCGGC AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9 GA223 GCTGACCA 217gscsusGACCACCCCUGCCAGGUG 395 SpCas9 CCCCTGCC UUUUAGAGCUAGAAAUAGCAA AGGTGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA224 GGCCGCTG 218 gsgscsCGCUGACCACCCCUGCCG 396 SpCas9 ACCACCCCUUUUAGAGCUAGAAAUAGCAA TGCC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA225 GGCATCGT 219 gsgscsAUCGUCCCGGAAGUUGCG397 SpCas9 CCCGGAA UUUUAGAGCUAGAAAUAGCAA GTTGC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA226 GGCTTTTC 220gsgscsUUUUCCGAAUAAACUCCG 398 SpCas9 CGAATAA UUUUAGAGCUAGAAAUAGCAA ACTCCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA227 GTCCCGGA 221 gsuscsCCGGAAGUUGCCGGCAGG 399 SpCas9 AGTTGCCGUUUUAGAGCUAGAAAUAGCAA GCAG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA228 CGGCTGTA 222 csgsgsCUGUACCCACCCGCCAGG400 SpCas9 CCCACCCG UUUUAGAGCUAGAAAUAGCAA CCAG GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA229 GTCGTGCT 223gsuscsGUGCUGGUCACCGCUGCG 401 SpCas9 GGTCACCG UUUUAGAGCUAGAAAUAGCAA CTGCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA230 AGTAGAG 224 asgsusAGAGGCAGGCAUCGUCCG 402 SpCas9 GCAGGCATUUUUAGAGCUAGAAAUAGCAA CGTCC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA231 TCCGAATA 225 uscscsGAAUAAACUCCAGGCCUG403 SpCas9 AACTCCAG UUUUAGAGCUAGAAAUAGCAA GCCT GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA232 GTTTATTC 226gsususUAUUCGGAAAAGCCAGC 404 SpCas9 GGAAAAG GUUUUAGAGCUAGAAAUAGCA CCAGCAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hPCSK9GA233 GCGGCTGT 227 gscsgsGCUGUACCCACCCGCCAG 405 SpCas9 ACCCACCCUUUUAGAGCUAGAAAUAGCAA GCCA GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA235 CACCGCTG 228 csascsCGCUGCCGGCAACUUCCG406 SpCas9 CCGGCAAC UUUUAGAGCUAGAAAUAGCAA TTCC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA236 AGCCCTCG 229asgscsCCUCGCCAGGCGCUGGCG 407 SpCas9 CCAGGCGC UUUUAGAGCUAGAAAUAGCAA TGGCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA237 CAACGCCG 230 csasasCGCCGCCUGCCAGCGCCG 408 SpCas9 CCTGCCAGUUUUAGAGCUAGAAAUAGCAA CGCC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA238 GCACGACC 231 gscsasCGACCCCAGCCCUCGCCG409 SpCas9 CCAGCCCT UUUUAGAGCUAGAAAUAGCAA CGCC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA239 CCGCCTGC 232cscsgsCCUGCCAGCGCCUGGCGG 410 SpCas9 CAGCGCCT UUUUAGAGCUAGAAAUAGCAA GGCGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA240 TGCCAGCG 233 usgscsCAGCGCCUGGCGAGGGCG 411 SpCas9 CCTGGCGAUUUUAGAGCUAGAAAUAGCAA GGGC GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA241 TGCTGCTG 234 usgscsUGCUGCCCCUGGCGGGUG412 SpCas9 CCCCTGGC UUUUAGAGCUAGAAAUAGCAA GGGT GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA242 TCACCGCT 235uscsasCCGCUGCCGGCAACUUCG 413 SpCas9 GCCGGCA UUUUAGAGCUAGAAAUAGCAA ACTTCGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA243 GGCGAGG 236 gsgscsGAGGGCUGGGGUCGUGC 414 SpCas9 GCTGGGGTGUUUUAGAGCUAGAAAUAGCA CGTGC AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA244 TTCCGAAT 237 ususcsCGAAUAAACUCCAGGCCG415 SpCas9 AAACTCCA UUUUAGAGCUAGAAAUAGCAA GGCC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA245 GTGCTGCT 238gsusgsCUGCUGCCCCUGGCGGGG 416 SpCas9 GCCCCTGG UUUUAGAGCUAGAAAUAGCAA CGGGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9GA246 GCCAGCGC 239 gscscsAGCGCCUGGCGAGGGCUG 417 SpCas9 CTGGCGAGUUUUAGAGCUAGAAAUAGCAA GGCT GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA247 CCTCGCCA 240 cscsusCGCCAGGCGCUGGCAGGG418 SpCas9 GGCGCTGG UUUUAGAGCUAGAAAUAGCAA CAGG GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA248 GGTGCTAG 66gsgsusG(cPACE)UAGCCUUGCGU 419 SpCas9 CCTTGCGT UCCGGUUUUAGAgcuagaaauagcATCCG AGUUAAAAUAAGGCUAGUCCG UUAUCAacuugaaaaaguggcaccgaguc ggugcusususuhcPCSK9 GA249 GGTGCTAG 66 gsgsusGCUAGCC(uPACE)UGCGU 420 SpCas9 CCTTGCGTUCCGGUUUUAGAgcuagaaauagcA TCCG AGUUAAAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgaguc ggugcusususu hcPCSK9 GA250 GGTGCTAG 66gsgsusGcUAGCCUUGCGUUCCGG 253 SpCas9 CCTTGCGT UUUUAGAgcuagaaauagcAAGUUATCCG AAAUAAGGCUAGUCCGUUAUC Aacuugaaaaaguggcaccgagucggugcusu susu hcPCSK9GA251 GGTGCTAG 66 gsgsusGCUAGCCuUGCGUUCCGG 253 SpCas9 CCTTGCGTUUUUAGAgcuagaaauagcAAGUUA TCCG AAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusu susu hcPCSK9 GA253 GGTGCTAG 66gsgsusG(cPACE)UAGCCUUGCGU 419 SpCas9 CCTTGCGTUCCGgUUUUAGagcuagaaauagcaaG TCCG UUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusu susu hcPCSK9 GA254 GGTGCTAG 66gsgsusGCUAGCC(uPACE)UGCGU 420 SpCas9 CCTTGCGTUCCGgUUUUAGagcuagaaauagcaaG TCCG UUaAaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusu susu mPCSK9 GA255 CCCATACC 69cscscsAUACCUUGGAGCAACGGg 421 ABE TTGGAGCA UUUUAGagcuaGaaauagcaaGUUaAaACGG AuAaggCUaGUCcGUUAucAAcuuG aaaaaguGgcaccgAgUCggugcusususu mPCSK9GA256 CCCATACC 69 cscscsAUACCUUGGAGCAACGGg 421 ABE TTGGAGCAUUUUAGagcuagaaauagcaaGUUaAa ACGG AuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu mPCSK9 GA257 CCCATACC 69cscscsAUACCUUGGAGCAACGGg 421 ABE TTGGAGCA UUUUAGagcuaGaaauagcaaGUUaAaACGG AuAaggcuaGUccGUUAucAAcuuGa aaaagugGcaccgagucggugcusususu mANGPTL3GA258 GAGATACC 241 gsasgsAUACCUGAGUAACUUUCg 422 ABE TGAGTAACUUUUAGagcuaGaaauagcaaGUUaAa TTTC AuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcusususu mANGPTL3 GA259 GAGATACC 241gsasgsAUACCUGAGUAACUUUCg 422 ABE TGAGTAAC UUUUAGagcuagaaauagcaaGUUaAaTTTC AuAaggcuaGUccGUUAucAAcuugaa aaagugGcaccgagucggugcusususu mANGPTL3GA260 GAGATACC 241 gsasgsAUACCUGAGUAACUUUCg 422 ABE TGAGTAACUUUUAGagcuaGaaauagcaaGUUaAa TTTC AuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu hcANGPTL3 GA276 ACGTGGG 242ascsgsUGGGAGAACUACAAAUA 423 ABE AGAACTAC GUUUUAGAGCUAGAAAUAGCA AAATAAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususuhcANGPTL3 GA277 CGATGTTG 243 csgsasUGUUGAAUUAAUGUCCA 424 ABE AATTAATGGUUUUAGAGCUAGAAAUAGCA TCCA AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcANGPTL3 GA278 CACAAAA 244csascsAAAACUUCAAUGAAACGG 425 ABE CTTCAATG UUUUAGAGCUAGAAAUAGCAA AAACGGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususuhcANGPTL3 GA279 TACGAATT 245 usascsGAAUUGAGUUGGAAGAC 426 ABE GAGTTGGAGUUUUAGAGCUAGAAAUAGCA AGAC AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcANGPTL3 GA284 CTATGGAG 246csusasUGGAGUAUAUCUUCUCU 427 ABE TATATCTT GUUUUAGAGCUAGAAAUAGCA CTCTAGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUsususu hcPCSK9GA343 CCCGCACC 13 cscscsGCACCUUGGCGCAGCGGg 9 ABE TTGGCGCAUUUUAGAGCUAGAAAUAGCAA GCGG GUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu cANGPTL3 GA344 AAGATACC 14 asasgsAUACCUGAAUAACUCUCG257 ABE TGAATAAC UUUUAGAGCUAGAAAUAGCAA TCTC GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususu hcPCSK9 GA346 CCCGCACC 13cscscsGCACCUUGGCGCAGCGGg 9 ABE TTGGCGCA UUUUAGagcuaGaaauagcaaGUUaAa GCGGAuAaggcuaGUccGUUAucAAcuuGa aaaagugGcaccgagucggugcusususu cANGPTL3 GA347AAGATACC 14 asasgsAUACCUGAAUAACUCUCg 257 ABE TGAATAACUUUUAGagcuaGaaauagcaaGUUaAa TCTC AuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu mANGPTL3 GA349 GAGATACC 241gsasgsAUACCUGAGUAACUUUC 422 ABE TGAGTAAC GUUUUAGAgcuagaaauagcAAGUU TTTCAAAAUAAGGCUAGUCCGUUAU CAacuugaaaaaguggcaccgagucggugcu sususu mANGPTL3GA353 GAGATACC 241 gsasgsAUACCUGAGUAACUUUC 422 ABE TGAGTAACGUUUUAGAGCUAGAAAUAGCA TTTC AGUUAAAAUAAGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsususu hcPCSK9 GA375 CCCGCACC 13 cscscsGCACCUUGGCGCAGCGGg428 ABE TTGGCGCA UUUUAGagcuagaaauagcaaGUUaAa GCGGAuAaggcuaGUccGUUAacAAcgggaa accgugGcaccgagucggugcusususu hcPCSK9 GA376CCCGCACC 13 cscscsdGCdACdCUdUGdGCdGCdA 429 ABE TTGGCGCAGdCGdGgUUUUAGagcuaGaaauagc GCGG aaGUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugc usususu hcPCSK9 GA377 CCCGCACC 13cscscsGdCAdCCdUUdGGdCGdCAd 430 ABE TTGGCGCA GCdGGgUUUUAGagcuaGaaauagcaaGCGG GUUaAaAuAaggcuaGUccGUUAuc AAcuuGaaaaagugGcaccgagucggugcu sususuhcPCSK9 GA380 CCCGCACC 13 cscscsGdCACCUUGGCGdCAGCG 431 ABE TTGGCGCAGgUUUUAGagcuaGaaauagcaaGUUa GCGG AaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu hcPCSK9 GA381 CCCGCACC 13cscscsGCdACCUUGGCGCdAGCG 432 ABE TTGGCGCA GgUUUUAGagcuaGaaauagcaaGUUaGCGG AaAuAaggcuaGUccGUUAucAAcuu GaaaaagugGcaccgagucggugcusususu hcPCSK9GA382 CCCGCACC 13 cscscsGCACCUdUGGdCGCAGdCG 433 ABE TTGGCGCAGgUUUUAGagcuaGaaauagcaaGUUa GCGG AaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu hcPCSK9 GA383 CCCGCACC 13cscscsGCACCUUdGGCdGCAGCdG 434 ABE TTGGCGCA GgUUUUAGagcuaGaaauagcaaGUUaGCGG AaAuAaggcuaGUccGUUAucAAcuu GaaaaagugGcaccgagucggugcusususu hcPCSK9GA384 CCCGCACC 13 cscscsdGdCdACCdUdUGGdCGCAd 435 ABE TTGGCGCAGCGdGgUUUUAGagcuaGaaauagcaa GCGG GUUaAaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcu sususu hcPCSK9 GA385 CCCGCACC 13cscscsGCACCUUGGCGCAGCGGg 11 ABE TTGGCGCA UUUUAGagcuagaaauagcaaGUUaAaGCGG AuAaggcuaGUccGUUAucAAcuugaa aaagugGcaccgagucggugcusususuuuu hcPCSK9GA386 CCCGCACC 13 cscscsGCACCUUGGCGCAGCGGg 11 ABE TTGGCGCAUUUUAGagcuagaaauagcaaGUUaAa GCGG AuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuUu hcPCSK9 GA387 CCCGCACC 13cscscsGCACCUUGGCGCAGCGgU 436 ABE TTGGCGCA UUUAGagcuaGaaauagcaaGUUaAaAGCGG uAaggcuaGUccGUUAucAAcuuGaaa aagugGcaccgagucggugcuususuuuu hcPCSK9GA388 CCCGCACC 13 cscscsGCACCUUGGCGCAGCGGg 9 ABE TTGGCGCAUUUUAGagcuagaaauagcaaGUUaAa GCGG AuAaggcuaGUCcGUUAucAAcuugaaaaaguggcaccgagucggugcusususu hcPCSK9 GA389 CCCGCACC 13cscscsGCACCUUGGCGCAGCGGg 9 ABE TTGGCGCA UUUUAGagcuagaaauagcaaGUUaAa GCGGAuAaggcuaGuCcGUUaucAAcuugaa aaaguggcaccgagucggugcusususu hcPCSK9 GA391CCGCACCT 247 cscsGCACCUUGGCGCAGCGGgU 437 ABE TGGCGCAGUUUAGagcuaGaaauagcaaGUUaAaA CGG uAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu hANGPTL3 GA441 AAGATACC 15asasgsAUACCUGAAUAACCCUCg 59 ABE TGAATAAC UUUUAGagcuaGaaauagcaaGUUaAaCCTC AuAaggcuaGUccGUUAucAAcuuGa aaaagugGcaccgagucggugcusususu hANGPTL3GA442 AAGATACC 15 asasgsAUACCUGAAUAACCCUCg 438 ABE TGAATAACUUUUAGagcuaGaaauagcaaGUUaAa CCTC AuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususuUU u hANGPTL3 GA472 AAGATACC 15asasisAUACCUGAAUAACCCUCg 439 ABE TGAATAAC UUUUAGagcuaGaaauagcaaGUUaAaCCTC AuAaggcuaGUccGUUAucAAcuuGa aaaagugGcaccgagucggugcusususu hANGPTL3GA473 AAGATACC 15 asasisAUACCUIAAUAACCCUCgU 440 ABE TGAATAACUUUAGagcuaGaaauagcaaGUUaAaA CCTC uAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu hANGPTL3 GA474 AAGATACC 15asasgsAUACCUIAAUAACCCUCgU 441 ABE TGAATAAC UUUAGagcuaGaaauagcaaGUUaAaACCTC uAaggcuaGUccGUUAucAAcuuGaaa aagugGcaccgagucggugcusususu hANGPTL3GA475 AGATACCT 248 asgsasUACCUGAAUAACCCUCgU 442 ABE GAATAACCUUUAGagcuaGaaauagcaaGUUaAaA CTC uAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu hANGPTL3 GA476 GATACCTG 249gsasUACCUGAAUAACCCUCgUU 443 ABE AATAACCC UUAGagcuaGaaauagcaaGUUaAaAu TCAaggcuaGUccGUUAucAAcuuGaaaa agugGcaccgagucggugcusususu hANGPTL3 GA477ATACCTGA 250 asusACCUGAAUAACCCUCgUUU 444 ABE ATAACCCTUAGagcuaGaaauagcaaGUUaAaAuA C aggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu hANGPTL3 GA547 AAGATACC 14asasgsAUACCUGAAUAACUCUCg 445 ABE TGAATAAC UUUUAGagcuaGaaauagcaaGUUaAaTCTC AuAaggcuaGUccGUUAucAAcuuGa aaaaguggcaccgagucggugcusususuUU u mPCSK9GA010 GGCTGATG 251 5′gsgscsUGAUGAGGCCGCACAU 446 SpCas9 AGGCCGCGGUUUUAGAgcuagaaauagcAAGU ACATG UAAAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugc usususu-3′ mPCSK9 GA011 GGCTGATG 2515′gsgscsUGAUGAGGCCGCACAU 446 SpCas9 AGGCCGC GGUUUUAGAgcuagaaauagcAAGUACATG UAAAAUAAGGCUAGUCCGUUA UCAacuugaaaaaguggcaccgagucggugc usususu-3′hcANGPTL3 GA016 GGCCTCCT 107 5′gsgscsCUCCUUCAGUUGGGAC 285 SpCas9TCAGTTGG AGUUUUAGAgcuagaaauagcAAGU GACA UAAAAUAAGGCUAGUCCGUUAUCAacuugaaaaaguggcaccgagucggugc usususu-3′ hcANGPTL3 GA017 GCCAATGG 1095′gscscsAAUGGCCUCCUUCAGU 287 SpCas9 CCTCCTTC UGUUUUAGAgcuagaaauagcAAGUAGTT UAAAAUAAGGCUAGUCCGUUA UCAacuugaaaaaguggcaccgagucggugc usususu-3′hcPCSK9 GA395 TCAGCTCC 161 5′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuaGaaauagcaaGUUaA CTGG aAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususuuu u-3′ hcPCSK9 GA396 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 448 SpCas9 AGGCGGTCgUUUUAGagcuaGaaauagcaaGUUsa CTGG AaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu uuu-3′ hcPCSK9 GA397 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 449 SpCas9 AGGCGGTCgUUUUAGagcuaGaaauagcaaGUUaA CTGG aAuAaggcuaGUccGUUsAucAAcuuGaaaaagugGcaccgagucggugcusususu uuu-3′ hcPCSK9 GA398 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuagaaauagcaaGUUaA CTGG aAuAaggcuaGUCcGUUAucAAcuugaaaaaguggcaccgagucggugcusususuuu u-3′ hcPCSK9 GA399 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuagaaauagcaaGUUaA CTGG aAuAaggcuaGuCcGUUaucAAcuugaaaaaguggcaccgagucggugcusususuuu u-3′ hcPCSK9 GA400 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUuagagcuagaaauagcaaGUUaAa CTGG AuaaggcuaGuccgUUaucaacuugaaaaaguggcaccgagucggugcusususuuuu-3′ hcPCSK9 GA401 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuagaaauagcaaGUUaA CTGG aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuu u-3′ hcPCSK9 GA402 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuaGaaauagcaaGUUaA CTGG aAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususuu Uu-3′ hcPCSK9 GA403 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 448 SpCas9 AGGCGGTCgUUUUAGagcuaGaaauagcaaGUUsa CTGG AaAuAaggcuaGUccGUUAucAAcuuGaaaaagugGcaccgagucggugcusususu uUu-3′ hcPCSK9 GA404 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 449 SpCas9 AGGCGGTCgUUUUAGagcuaGaaauagcaaGUUaA CTGG aAuAaggcuaGUccGUUsAucAAcuuGaaaaagugGcaccgagucggugcusususu uUu-3′ hcPCSK9 GA405 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuagaaauagcaaGUUaA CTGG aAuAaggcuaGUCcGUUAucAAcuugaaaaaguggcaccgagucggugcusususuuU u-3′ hcPCSK9 GA406 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuagaaauagcaaGUUaA CTGG aAuAaggcuaGuCcGUUaucAAcuugaaaaaguggcaccgagucggugcusususuuU u-3′ hcPCSK9 GA407 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUuagagcuagaaauagcaaGUUaAa CTGG AuaaggcuaGuccgUUaucaacuugaaaaaguggcaccgagucggugcusususuuUu-3′ hcPCSK9 GA408 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 447 SpCas9 AGGCGGTCgUUUUAGagcuagaaauagcaaGUUaA CTGG aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuuU u-3′ hcPCSK9 GA409 TCAGCTCC 1615′uscsasG(C- 450 SpCas9 AGGCGGTC PACE)UCCAGGCGGUCCUGGgU CTGGUUUAGagcuagaaauagcaaGUUaAaA uAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ hcPCSK9 GA410 TCAGCTCC 1615′uscsasGCUCCAG(G- 451 SpCas9 AGGCGGTC PACE)CGGUCCUGGgUUUUAGag CTGGcuagaaauagcaaGUUaAaAuAaggcuaG UccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ hcPCSK9 GA439 CCCGCACC 13 5cscscsGCACCUUGGCGCAGCGG11 ABE TTGGCGCA gUUUUAGagcuaGaaauagcaaGUUaA GCGGaAuAaggcuaGUccGUUAucAAcuuG aaaaagugGcaccgagucggugcusususuU Uu-3′ hcPCSK9GA440 CCCGCACC 13 5′cscscsGCACCUUGGCGCAGCGG 11 ABE TTGGCGCAgUUUUAGagcuagaaauagcaaGUUaA GCGG aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususuUU u-3′ hcPCSK9 GA234 CGCCTGCC 252csgscsCUGCCAGCGCCUGGCGAG 452 SpCas9 AGCGCCTG UUUUAGAGCUAGAAAUAGCAA GCGAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUsususuhcANGPTL3 GA261 TGAAGAA 126 5′usgsasAGAAAGGGAGUAGUUC 304 SpCas9 AGGGAGTUgUUUUAGagcuagaaauagcaaGUUa AGTTCT AaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcususus u-3′ hcANGPTL3 GA262 CATTATAT 1435′csasusUAUAUUCAGGUAGUCC 321 SpCas9 TCAGGTAG AgUUUUAGagcuagaaauagcaaGUUaTCCA AaAuAaggcuaGUccGUUAucAAcuu gaaaaagugGcaccgagucggugcususus u-3′hcANGPTL3 GA263 ACAAAACT 146 5′ascsasAAACUUCAAUGAAACG 324 SpCas9TCAATGAA UgUUUUAGagcuagaaauagcaaGUUa ACGT AaAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcususus u-3′ hcANGPTL3 GA264 GCCAATGG 1095′gscscsAAUGGCCUCCUUCAGU 287 SpCas9 CCTCCTTC UgUUUUAGagcuagaaauagcaaGUUaAGTT AaAuAaggcuaGUccGUUAucAAcuu gaaaaagugGcaccgagucggugcususus u-3′hcANGPTL3 GA265 GCCAATGG 109 5′gscscsAAUGGCCUCCUUCAGU 287 SpCas9CCTCCTTC UgUUUUAGagcuaGaaauagcaaGUUa AGTT AaAuAaggCUaGUCcGUUAucAAcuuGaaaaaguGgcaccgAgUCggugcususu su-3′ hcPCSK9 GA266 TCAGCTCC 1615′uscsasGCUCCAGGCGGUCCUGG 339 SpCas9 AGGCGGTCgUUUUAGagcuagaaauagcaaGUUaA CTGG aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ hcPCSK9 GA267 GCCCCATG 1855′gscscsCCAUGUCGACUACAUCG 363 SpCas9 TCGACTACgUUUUAGagcuagaaauagcaaGUUaA ATCG aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ hcPCSK9 GA268 GGGGCTG 1875′gsgsgsGCUGGUAUUCAUCCGC 365 SpCas9 GTATTCAT UgUUUUAGagcuagaaauagcaaGUUaCCGCC AaAuAaggcuaGUccGUUAucAAcuu gaaaaagugGcaccgagucggugcususus u-3′hcPCSK9 GA269 CTAGGAG 194 5′csusasGGAGAUACACCUCCACC 372 SpCas9 ATACACCTgUUUUAGagcuagaaauagcaaGUUaA CCACC aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ hcPCSK9 GA270 CTGCGCGT 2045′csusgsCGCGUGCUCAACUGCCA 382 SpCas9 GCTCAACTgUUUUAGagcuagaaauagcaaGUUaA GCCA aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ hcPCSK9 GA271 CGGGATGC 2065′csgsgsGAUGCCGGCGUGGCCA 384 SpCas9 CGGCGTGG AgUUUUAGagcuagaaauagcaaGUUaCCAA AaAuAaggcuaGUccGUUAucAAcuu gaaaaagugGcaccgagucggugcususus u-3′hcPCSK9 GA272 CGCTGACC 212 5′csgscsUGACCACCCCUGCCAGG 390 SpCas9 ACCCCTGCgUUUUAGagcuagaaauagcaaGUUaA CAGG aAuAaggcuaGUccGUUAucAAcuugaaaaagugGcaccgagucggugcusususu-3′ mPCSK9 GA292 cccatacC 695′cscscsAUACCUUGGAGCAACG 421 ABE TTGGAGCA GGUUUUAGAGCUAGAAAUAGC ACGGAAGUUAAAAUAAGGCUAGUCC GUUAUCAACUUGAAAAAGUGG CACCGAGUCGGUGCUsususu-3′

As used in Table 1 above, Table 23, and Table 24, in the Protospacer:uppercase nucleotides (A, G, C, I and T) indicate2′-deoxyribonucleotides, adenine, guanine, cytosine, inosine, andthymine, respectively; in Guide RNA sequence: uppercase nucleotides (A,C, G, I and U) indicate ribonucleotides, adenine, guanine, cytosine,inosine, and uracil, respectively and lowercase nucleotides (a, g, c, iand u) indicate 2′-O-methylribonucleotide (2′-OMe) unless otherwisespecified. It is understood that the DNA protospacer is converted to RNAor RNA-equivalent in the guide RNA design except when othermodifications including 2′-deoxyribonucleotides are introduced into thespacer section of the single guide RNA; s: phosphorothioate (PS),X=ribonebularine; x=2′-O-methylnebularine; dX=2′-deoxynebularine;5′-NNN-3′ indicates uniform A, C, G, I, U, dA, dG, dC, dI, T, X, x, dX,and combinations thereof. 5′-nnn-3′ indicates uniform a, c, g, i, u, dA,dG, dC or T, x, dX, and combinations thereof. mANGPTL3: mouse ANGPTL3;hANGPTL3: human ANGPTL3; cANGPTL3: cynomolgus ANGPTL3; mcANGPTL3: mouse,cynomolgus cross-reactive ANGPTL3; hcANGPTL3: human, cynomolguscross-reactive ANGPTL3; mPCSK9: mouse PCSK9; hPCSK9: human PCSK9;cPCSK9: cynomolgus PCSK9; mcPCSK9: mouse, cynomolgus cross-reactivePCSK9; hcPCSK9: human, cynomolgus cross-reactive PCSK9; hcAPOC3: human,cynomolgus cross-reactive APOC3. As disclosed herein, the nucleotidesequences and modification patterns encompass all length, structure, andtype of RNAs or fragments thereof, CRISPR guide RNAs, e.g. sgRNAs, dualguide RNAs, or mRNAs. For example, nucleotide sequences and modificationpatterns as described in the Table above may indicate RNA sequences andmodification patterns in a single guide RNA, a dual guide RNA, anuclease mRNA, or any fragment or segment thereof. In Table 23, u′indicates N¹-methylpseudouridine.

Example 2. PCSK9 and ANGPTL3 Cas9 Editing In Vitro

In set of experiments, PCSK9 gRNAs were co-transfected with anequivalent amount (1:1 ratio by weight) of in vitro transcribedcommercially available SpCas9 mRNA MS002 purchased from TriLinkBiotechnologies into primary human hepatocytes at 2500, 500, or 100ng/RNA/mL and processed as described in detailed methods. Extractedgenomic DNA was analyzed for gene editing at the target site withnext-generation sequencing of PCR amplicons generated around the targetsites. Samples were prepared using the Nextera XT DNA librarypreparation kit (Illumina) according to the manufacturer's protocol.Briefly, two rounds of PCR were performed first to amplify the region ofinterest and second to add DNA sequences required for next generationsequencing and sample identification to the initial product. The finalamplicon was sequenced on the Illumina MiSeq instrument according to themanufacturer's protocol. A wide range of editing activities (Table 2)were observed. In a second set of experiments, ANGPTL3 gRNAs wereco-transfected with an equivalent amount of in vitro transcribed SpCas9mRNA (1:1 ratio by weight) into primary human hepatocytes and processedand analyzed similarly (Table 3).

TABLE 2 PCSK9 gRNA/SpCas9 editing in primary hepatocytes SEQHuman Primary Cyno Primary Hepatocyte Protospacer IDHepatocyte Editing % Editing % gRNA (5′-3′) NO: 2500 500 100 2500 500100 GA15 GGTGCTAGCCT 66 26.34 6.22 1.48 33.94 14.31 2.34 6 TGCGTTCCGGA15 GCCGTCCTCCT 151 10.03 2.82 0.62 13.78 2.93 0.78 7 CGGAACGCA GA15GCTAGCCTTGC 152 37.94 6.55 2 18.27 4.42 1.04 8 GTTCCGAGG GA15GCGTTCCGAGG 153 14.68 3.74 1.35 12.21 3.69 1.19 9 AGGACGGCC GA16GCCTTGCGTTC 154 23.3 5.58 1.19 23.15 6.60 1.38 0 CGAGGAGGA GA16GGACGAGGAC 155 35.08 8.74 3.65 48.24 14.98 4.52 1 GGCGACTACG GA16GGACGGCGAC 156 42.8 12.97 2.14 28.26 11.18 1.62 2 TACGAGGAGC GA16CGTCCTCGTCC 157 29.32 9.2 2.31 49.50 17.69 5.99 3 TCCTGCGCA GA16GTCCTCGTCCT 158 25.77 11.12 2.68 34.89 13.96 2.75 4 CCTGCGCAC GA16CCGTCAGCTCC 159 40.27 22.3 4.99 35.57 18.00 5.43 5 AGGCGGTCC GA16CGCCCGTGCGC 160 36.89 10.99 2.89 44.77 14.39 3.93 6 AGGAGGACG GA16TCAGCTCCAGG 161 53.71 25.87 8.2 66.07 35.98 11.98 7 CGGTCCTGG GA16CTTGGCGCAGC 162 54.99 25.77 ND 41.49 15.90 4.75 8 GGTGGAAGG GA16CGTGCGCAGG 163 46.65 15.15 5.49 42.38 18.28 3.45 9 AGGACGAGGA GA17CCAGGACCGC 164 31.35 15.84 2.9 23.17 11.48 4.03 0 CTGGAGCTGA GA17TCTTGGTGAGG 165 31.75 9.38 2.37 27.79 9.06 1.29 1 TATCCCCGG GA17CAGTGCGCTCT 166 14.01 3.52 0.32 15.58 2.11 0.51 2 GACTGCGAG GA17CTTGGTGAGGT 167 23.38 3.94 0.69 35.03 8.70 2.06 3 ATCCCCGGC GA17GGATCTTGGTG 168 14.74 5.24 1.18 18.65 4.88 1.60 4 AGGTATCCC GA17GAAGATGAGT 169 24.81 7.53 1.55 44.77 12.37 1.45 5 GGCGACCTGC GA17AGCACCACCA 170 4.98 1.26 0.36 22.65 8.11 2.11 6 CGTAGGTGCC GA17GGTCGCCACTC 171 12.57 2.67 0.65 15.91 4.68 0.38 7 ATCTTCACC GA17CTCCTTCAGCA 172 18.29 5.43 1.26 25.22 5.78 2.21 8 CCACCACGT GA17GAGTGGCGAC 173 21.87 6 1.29 12.94 4.21 1.06 9 CTGCTGGAGC GA18GCGCACTGCCC 174 17.16 2.22 0.22 14.12 2.45 0.91 0 GCCGCCTGC GA18GGCTTCCTGGT 175 ND ND 28.87 45.12 15.43 3.66 1 GAAGATGAG GA18CACCTACGTGG 176 17.44 4.49 0.95 22.89 6.14 1.55 2 TGGTGCTGA GA18CTACGTGGTGG 177 32.35 9.89 2.21 39.56 15.99 2.87 3 TGCTGAAGG GA18ATGGAAGACA 178 29.55 8.29 2.65 31.87 10.54 2.90 4 TGCAGGATCT GA18AGACATGCAG 179 27.98 10.91 1.6 20.29 7.75 1.14 5 GATCTTGGTG GA18CTCATCTTCAC 180 12.01 3.57 0.79 33.48 7.59 1.96 6 CAGGAAGCC GA18CTCCTCGATGT 181 ND 6.47 2.44 29.24 12.30 3.77 7 AGTCGACAT GA18TATTCATCCGC 182 41.02 15.38 4.21 0.06 0.01 0.01 8 CCGGTACCG GA18TCCTCGATGTA 183 31.23 8.94 1.76 41.70 15.58 3.30 9 GTCGACATG GA19CCTCCTCGATG 184 25.39 9.73 2.78 26.78 5.53 1.17 0 TAGTCGACA GA19GCCCCATGTCG 185 34.6 12.2 3 51.79 26.79 5.46 1 ACTACATCG GA19CCATGTCGACT 186 16.7 6.02 1.14 10.26 1.50 0.60 2 ACATCGAGG GA19GGGGCTGGTAT 187 35.77 15.68 3.92 52.89 32.02 8.70 3 TCATCCGCC GA19GTCGACATGG 188 32.97 12.39 2.32 30.91 9.49 2.38 4 GGCAACTTCA GA19ACCACCGGGA 189 12.57 13.3 3.58 18.78 4.77 0.69 5 AATCGAGGGC GA19GAGTGACCAC 190 17.19 2.9 0.75 27.79 3.4 1.09 6 CGGGAAATCG GA19AGTGACCACC 191 15.03 3.29 1.22 18.21 3.89 0.9 7 GGGAAATCGA GA19CCACCGGGAA 192 30.93 10.58 0.04 49 16.15 3.53 8 ATCGAGGGCA GA19GAAGCGGGTC 193 29.95 6.89 1.17 45.32 15.11 3.07 9 CCGTCCTCCT GA20CTAGGAGATA 194 5.31 8.68 3.8 54.36 20.51 5.15 0 CACCTCCACC GA20CAGCATACAG 195 31.51 12.92 3.29 44.01 14.37 4.33 1 AGTGACCACC GA20AAGCGGGTCC 196 34.75 10.75 2.54 31.13 8.91 1.54 2 CGTCCTCCTC GA20TGACCCTGCCC 197 37.02 12.76 2.57 32.85 10.49 3.66 3 TCGATTTCC GA20CACTCTGTATG 198 24.98 8.98 1.27 19.37 5.27 0.91 4 CTGGTGTCT GA20CCCTGCCCTCG 199 38.04 12.42 3.74 36.43 12.04 3.01 5 ATTTCCCGG GA20CCGGTGGTCAC 200 20.73 2.7 0.85 16.9 2.73 0.84 6 TCTGTATGC GA20GGAAATCGAG 201 20.79 3.46 0.84 19.16 2.72 1.05 7 GGCAGGGTCA GA20CCAGCATACA 202 25.76 5.72 1.38 25.07 6.01 1.15 8 GAGTGACCAC GA20CTTGGCAGTTG 203 33.23 8.76 0.02 40.78 13.96 3.59 9 AGCACGCGC GA21CTGCGCGTGCT 204 14.3 5.86 0.04 54.88 19.72 4.5 0 CAACTGCCA GA21TGCGCGTGCTC 205 38.07 10.29 1.31 28.63 8.3 1.01 1 AACTGCCAA GA21CGGGATGCCG 206 12.45 5.59 1.17 54.89 16.53 1.96 2 GCGTGGCCAA GA21CGTGCTCAACT 207 24.28 8.77 2.17 45.22 16.35 2.73 3 GCCAAGGGA GA21CCTTGGCCACG 208 22.92 6.28 1.94 50.35 14.84 3.06 4 CCGGCATCC GA21CAGCGGCCGG 209 4.94 1.01 0.13 7.52 1.07 5 GATGCCGGCG GA21 CCGGGATGCC 21014.65 3.7 0.95 37.79 4.97 1.77 6 GGCGTGGCCA GA21 GTGGTCAGCG 211 15.312.75 0.77 39.21 10.2 2.6 7 GCCGGGATGC GA21 CGCTGACCACC 212 31.71 8.963.81 58.66 19.95 5.42 8 CCTGCCAGG GA21 GGCAGGGGTG 213 15.97 4.57 1.2848.67 11.89 0.35 9 GTCAGCGGCC GA22 GTGCTCAACTG 214 21.87 9.12 2.17 45.1712.39 3.13 0 CCAAGGGAA GA22 TCATGGCACCC 215 29.35 7.48 1.11 57.4 20.272.82 1 ACCTGGCAG GA22 TGGCAGGGGT 216 2.04 0.66 0.5 16.7 3.32 0.88 2GGTCAGCGGC GA22 GCTGACCACCC 217 17.5 7.28 1.44 57.58 21.4 3.46 3CTGCCAGGT GA22 GGCCGCTGACC 218 13.02 2.19 1.01 47.5 12.34 2.61 4ACCCCTGCC GA22 GGCATCGTCCC 219 18.82 6.39 0.89 8.57 3.11 2.03 5GGAAGTTGC GA22 GGCTTTTCCGA 220 7.61 2.99 0.4 ND ND ND 6 ATAAACTCC GA22GTCCCGGAAGT 221 32.09 8.29 0.02 32.41 4.58 0.56 7 TGCCGGCAG GA22CGGCTGTACCC 222 37.1 10.96 5.9 31.36 7.89 1.95 8 ACCCGCCAG GA22GTCGTGCTGGT 223 30.41 6.85 0.47 34.28 5.39 0.75 9 CACCGCTGC GA23AGTAGAGGCA 224 29.31 12.83 ND 35.86 ND ND 0 GGCATCGTCC GA23 TCCGAATAAAC225 ND ND ND ND ND ND 1 TCCAGGCCT GA23 GTTTATTCGGA 226 ND ND ND ND ND ND2 AAAGCCAGC GA23 GCGGCTGTACC 227 19.97 5.39 3.08 37.8 10.84 2.38 3CACCCGCCA GA23 CGCCTGCCAGC 252 5.45 2.29 0.06 6.44 1.53 0.21 4 GCCTGGCGAGA23 CACCGCTGCCG 228 ND ND ND 50.03 17.51 2.51 5 GCAACTTCC GA23AGCCCTCGCCA 229 22.41 3.13 6.92 37.44 11.62 2.31 6 GGCGCTGGC GA23CAACGCCGCCT 230 36.78 15.83 4.53 46.01 13.46 2.17 7 GCCAGCGCC GA23GCACGACCCC 231 ND ND ND 17.92 4.01 0.94 8 AGCCCTCGCC GA23 CCGCCTGCCAG232 3.49 1.73 0.06 4.55 0.55 0.02 9 CGCCTGGCG GA24 TGCCAGCGCCT 233 7.940.24 1.05 10.62 1.9 0.52 0 GGCGAGGGC GA24 TGCTGCTGCCC 234 5.39 1.49 0.6116.11 2.94 0.97 1 CTGGCGGGT GA24 TCACCGCTGCC 235 ND ND ND 31.71 7.131.79 2 GGCAACTTC GA24 GGCGAGGGCT 236 ND ND ND 10.18 1.84 0.51 3GGGGTCGTGC GA24 TTCCGAATAAA 237 ND ND ND 28.16 7.89 1.63 4 CTCCAGGCCGA24 GTGCTGCTGCC 238 2.9 2.97 1.68 7.07 0.88 0.19 5 CCTGGCGGG GA24GCCAGCGCCTG 239 17.95 2.17 0.08 24.63 5.78 1.11 6 GCGAGGGCT GA24CCTCGCCAGGC 240 6.76 0.17 0.25 11.48 2.62 0.71 7 GCTGGCAGG

TABLE 3 ANGPTL3 gRNA/SpCas9 editing in primary hepatocytes SEQHuman Primary Hepatocyte Cyno Primary Hepatocyte Protospacer IDEditing % Editing % gRNA (5′-3′) NO: 500 ng/mL 100 ng/mL 500 ng/mL100 ng/mL GA107 AGCCAATGGCC 105 5.09 3.72 ND 32.58 TCCTTCAGT GA108TCCCAACTGAA 106 19.43 4.95 15.49 4.11 GGAGGCCAT GA109 GGCCTCCTTCA 10718.07 6.60 15.11 4.15 GTTGGGACA GA110 GACCATGTCCC 108 13.63 5.37 15.998.59 AACTGAAGG GA111 GCCAATGGCCT 109 24.08 10.72 28.98 22.61 CCTTCAGTTGA113 ATTCTGGAGGA 111 19.97 6.00 26.67 12.07 AATAACTAG GA115 AACATAGCAAA113 ND ND 25.51 12.58 TCTTGATTT GA116 GTAGAATTTTTT 114 16.33 3.78 19.8310.84 CTTCTAGG GA117 ACTACAAGTCA 115 16.55 7.08 26.11 10.01 AAAATGAAGGA122 CTTTTATTTGAC 120 13.82 4.45 16.76 7.28 TATGCTGT GA125 TGATGGTAAGA123 16.27 5.04 19.72 12.91 CACTTTGGT GA126 GGAGTAGTTCT 124 15.21 6.7317.57 8.29 TGGTGCTCT GA127 AACATGATGGT 125 18.74 5.77 33.67 19.89AAGACACTT GA128 TGAAGAAAGGG 126 25.39 10.29 32.55 0.19 AGTAGTTCT GA129AGTTCTTGGTGC 127 ND ND 20.06 5.36 TCTTGGCT GA130 ATGATGGTAAG 128 17.097.51 25.90 14.60 ACACTTTGG GA131 GAAGATAGAGA 129 18.64 ND 27.57 10.10AATTTCTGT GA132 GGAAGATAGAG 130 28.39 15.25 26 15 AAATTTCTG GA137TATCAGGTAAA 135 29.70 11.44 30.57 28.01 ACCTGTCTA GA141 TTGAGAGTTGC 13914.87 5.80 10.32 5.49 TGGGTCTGA GA142 TGAAAAACTTG 140 24.90 10.50 27.5920.09 AGAGTTGCT GA143 TTAATTCAACAT 141 19.36 9.30 21.16 14.84 CGAATAGAGA145 CATTATATTCAG 143 26.33 12.55 30.34 18.77 GTAGTCCA GA148ACAAAACTTCA 146 31.67 12.01 69.75 28.57 ATGAAACGT GA151 GAGAACTACAA 14931.88 10.56 38.91 11.32 ATATGGTTT

The modes of operation of Cas9, cytidine base editors (CBE), and adeninebase editors (ABE) respectively, along with relevant terminology used inthis application including “protospacer”, “PAM”, “spacer” areillustrated (FIGS. 1A-1C). For one of the guides that performed well,GA156, with spacer sequence 5′-GGTGCTAGCCTTGCGTTCCG-3′ (SEQ ID NO: 66),additional guides were synthesized with modifications to the tracrRNAsequence. Additionally, an X-ray crystal structure-guided approach wasused (FIG. 1D). Structure-guided spacer and tracr designs were informedby the crystal structures of S. pyogenes Cas9 in complex with sgRNA(Jiang et al., 2015, PDB ID 4ZT0) and S. pyogenes Cas9 in apre-catalytic ternary complex (Jiang et al., 2016, PDB ID 5F9R) (FIG. 2). Any positions in the crystal structures where there appeared to be ahydrogen bond between the 2′-OH of a given nucleotide and the Cas9protein (or another part of the sgRNA) were left unmodified.Additionally, any positions where a steric clash was predicted to occurbetween a 2′-OMe and the protein were left unmodified. At sites wherethe 2′-OH was solvent-exposed or otherwise distant from proteinresidues, a 2′-OMe substitution was made. In cases where the two crystalstructures disagreed, no modification was made. This strategy wasapplied to the entire sgRNA or just the tracr region. Exemplarystructures are shown in FIGS. 3-7 .

PCSK9 gRNAs were co-transfected with an equivalent amount of in vitrotranscribed SpCas9 mRNA MS002 (1:1 ratio by weight) into primary humanhepatocytes and processed as described in detailed methods. A wide rangeof editing activities were observed (Table 4; ND=Not determined). Theguides with the highest editing activity at five days post transfectionwere GA001, GA002, GA007, and GA008. The structure-guided 2′-OMe heavyguide RNA (gRNA) designs GA007 and GA008 showed improved editing overminimally modified control gRNA GA009.

TABLE 4 In vitro evaluation of modified gRNA via SpCas9 editing inprimary human hepatocytes gRNA % Editing, day 3 % Editing, day 5 GA00116 27 GA002 28 23 GA003 ND 7 GA004  7 12 GA005  1 15 GA006 ND 12 GA00730 27 GA008 20 28 GA009  4 19

Example 3. PCSK9 and ANGPTL3 Base Editing In Vitro

Editing of PCSK9 by ABE8.8 was observed in primary hepatocytes. PCSK9gRNAs were co-transfected with an equivalent amount of in vitrotranscribed ABE8.8 mRNA MA002 (1:1 ratio by weight) into primaryhepatocytes and processed as described in detailed methods. In one setof experiments, extracted genomic DNA was analyzed for base editing ofthe target splice site with next-generation sequencing of PCR ampliconsgenerated around the target sites. Samples were prepared using theNextera XT DNA library preparation kit (Illumina) according to themanufacturer's protocol. Briefly, two rounds of PCR were performed firstto amplify the region of interest and second to add DNA sequencesrequired for next generation sequencing and sample identification to theinitial product. The final amplicon was sequenced on the Illumina MiSeqinstrument according to the manufacturer's protocol. A wide range ofediting activities (Table 5; FIG. 8 ) were observed, with guides GA066,GA073, and GA074 performing the best.

TABLE 5 ABE8.8/PCSK9 gRNA editing in primary hepatocytes Human PrimaryCynomolgous Primary Hepatocytes- Hepatocytes- splice site splice siteediting % editing % 5000 2500 1250 5000 2500 1250 gRNA Species ng/mLng/mL ng/mL ng/mL ng/mL ng/mL GA066 Human/ 25.9 18.7 13.6 24.3 29.1 21.4Cyno GA072 Human/ 2.5 2.2 1.5 2.2 1.7 2.3 Cyno GA073 Human/ 25.4 17.511.2 29.2 23.2 24.7 Cyno GA074 Human/ 23.8 18.8 11.6 18.7 20.1 17.5 CynoGA075 Human/ 5.5 3.1 1.8 ND ND ND Cyno GA076 Human/ 4.9 3.9 1.6 ND ND NDCyno GA077 Human/ 6.0 5.7 2.8 5.4 5.7 4.1 Cyno GA078 Human/ 12.6 10.06.5 ND ND ND Cyno GA079 Human/ 6.5 4.9 2.2 0.5 0.5 0.6 Cyno GA080 Human/0.6 0.5 0.4 7.0 8.8 7.1 Cyno GA081 Human/ 8.3 6.1 4.3 0.3 0.3 0.3 CynoGA082 Human/ 9.8 9.6 5.6 ND ND ND Cyno GA083 Human/ 0.2 0.2 0.2 ND ND NDCyno GA084 Human/ 8.6 7.6 4.9 ND ND ND Cyno GA085 Human/ 2.4 1.8 1.2 NDND ND Cyno GA086 Human/ 5.7 4.9 2.9 ND ND ND Cyno GA087 Human/ 1.9 1.41.0 0.2 0.2 0.2 Cyno GA088 Human/ 7.2 6.2 3.6 24.8 23.0 17.0 Cyno GA089Human/ 18.1 14.1 5.4 ND ND ND Cyno GA090 Human/ 0.4 0.5 0.3 ND ND NDCyno

When considering the data from primary human hepatocytes,PCSK9-targeting gRNAs were identified that matched the following threeprotospacer sequences as having the best cross-species activity betweenboth primary human hepatocytes and primary cynomolgous hepatocytes:5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA066),5′-GCTTACCTGTCTGTGGAAGC-3′ (SEQ ID NO: 67) (GA073), and5′-TGCTTACCTGTCTGTGGAAG-3′ (SEQ ID NO: 68) (GA074). The GA066 sequencetargets the splice donor at the 5′ end of human PCSK9 intron 1 and ispredicted to result in an aberrant PCSK9 protein translated from exon 1,followed by several amino acids translated from the beginning of intron1 (read through from exon 1 into intron 1), followed by a premature stopcodon (FIG. 9 ). The GA073 and GA074 sequences each target the splicedonor at the 5′ end of human PCSK9 intron 4 and is predicted to resultin an aberrant PCSK9 protein translated from exons 1, 2, 3, and 4,followed by several amino acids translated from the beginning of intron4 (readthrough from exon 4 into intron 4), followed by a premature stopcodon. The predicted prematurely truncated protein in each of thesecases is likely to have complete loss of function as well as havingminimal immunogenicity due to the near-identical match to part of thenaturally occurring wild-type human PCSK9 amino acid sequence. The sameprotospacer sequence, 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13)(GA066), is found in the cynomolgus monkey genome, similarly targetingthe splice donor at the 5′ end of cyno PCSK9 intron 1.

Editing of ANGPTL3 by ABE8.8 was observed in primary hepatocytes.ANGPTL3 gRNAs were co-transfected with an equivalent amount of in vitrotranscribed ABE8.8 mRNA MA004 (1:1 ratio by weight) into primaryhepatocytes and processed as described in detailed methods. A humanANGPTL3-targeting gRNA matching the following protospacer sequence 5′-AAGATACCTGAATAACCCTC-3′ (SEQ ID NO: 15) (GA091) was identified. TheGA091 sequence targets the splice donor at the 5′ end of human ANGPTL3intron 6 and is predicted to result in an aberrant ANGPTL3 proteintranslated from exons 1, 2, 3, 4, 5, and 6, followed by several aminoacids translated from the beginning of intron 6 (readthrough from exon 6into intron 6), followed by a premature stop codon (FIG. 10 ). Notably,there is a naturally occurring rare human DNA variant that disrupts thesame splice donor and bears the designation rs398122985. A gRNA wassynthesized with the orthologous cynomolgous spacer sequence, differingby 1 nucleotide: 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14) (GA067) andbearing modest chemical modifications (Table 1). Both GA091 in humanprimary hepatocytes, and GA067 in cynomolgus primary hepatocytes, leadto substantial splice editing when co-transfected with ABE8.8 mRNA(protocol described in the detailed methods section) (Table 6). RNA wastransfected at 2500, 1250, 625, and 312.5 ng/test article/mL, with tworeplicates reported (rep 1 and rep 2).

TABLE 6 ABE8.8/ANGPTL3 gRNA editing in primary hepatocytes Human PrimaryHepatocytes-splice site editing % (Dose, Replicate #) 2500, 2500, 1250,1250, 625, 625, 312.5, 312.5, gRNA Species rep 1 rep 2 rep 1 rep 2 rep 1rep 2 rep 1 rep 2 GA091 Human 72.23 72.04 62.01 65.15 47.85 47.3 30.7129.62 GA092 Human/ 38.39 41.71 33.38 29.83 20.73 19.94 8.54 10.92 CynoGA093 Human 63.53 66.9 55.54 56.2 39.22 39.8 20.7 17.32 GA094 Human44.84 46.65 39.62 46.8 28.75 14.74 14.12 26.7 Cyno PrimaryHepatocytes-splice site editing (Dose, Replicate #) 2500, 2500, 1250,1250, 625, 625, 312.5, 312.5, rep 1 rep 2 rep 1 rep 2 rep 1 rep 2 rep 1rep 2 GA067 Cyno 66.06 64.84 64.92 63.78 53.56 53.66 37.05 39.36 GA092Human/ 45.76 43.27 43 44.28 32.42 32.56 25.72 19.87 Cyno

Another strategy by which adenine base editors might be used to disruptgene function is to introduce a missense mutation(s) into the codingregion of the gene that results in production of a less functional, ornon-functional protein. Guide RNAs targeting protospacers in ANGPTL3exons were assessed for base editing activity to introduce missensemutation(s). Each of the gRNAs with an equivalent amount of in vitrotranscribed ABE8.8 mRNA MA004 (1:1 ratio by weight) were co- transfectedin two replicates (rep 1 and rep 2) at 2500, 1250, 625, and 312.5ng/RNA/mL into primary human hepatocytes and processed as described indetailed methods. The resulting base editing efficiency, as well as onepotential amino acid substitution, is listed in Table 7.

In some embodiments, adenine base editors disrupts gene function byreplacing any one amino acid selected from A, R, N, D, C, Q, E, G, H, I,L, K, M, F, P, S, T, W, Y, and V with a non-identical amin acid A, R, N,D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In someembodiments, adenine base editors disrupt gene function by substitutingI with T or V. In some embodiments, adenine base editors disrupt genefunction by substituting D with G. In some embodiments, adenine baseeditors disrupt gene function by substituting E with G. In someembodiments, adenine base editors disrupt gene function by substitutingF with L or P. In some embodiments, adenine base editors disrupt genefunction by substituting H with R. In some embodiments, adenine baseeditors disrupt gene function by substituting L with P. In someembodiments, adenine base editors disrupt gene function by substitutingS with P. In some embodiments, adenine base editors disrupt genefunction by substituting M with T. In some embodiments, adenine baseeditors disrupt gene function by substituting N with G. In someembodiments, adenine base editors disrupt gene function by substitutingQ with R. In some embodiments, adenine base editors disrupt genefunction by substituting R with G. In some embodiments, adenine baseeditors disrupt gene function by substituting T with A. In someembodiments, adenine base editors disrupt gene function by substitutingV with A. In some embodiments, adenine base editors disrupt genefunction by substituting Y with C or H. In some embodiments, adeninebase editors disrupt gene function by substituting K with G. In someembodiments, adenine base editors disrupt gene function by introducingan amino acid substitute as listed in Table 7.

TABLE 7 ANGPTL3 gRNA ABE editing can introduce missense mutationsExample Amino Human Primary Hepatocytes-Editing % (Dose,Replicate #)Proto- SEQ Acid 2500, 2500, 1250, 1250, 6259 6259 312.5, 312.5, spacer ID Substi- rep rep rep rep rep rep rep rep gRNA (5′-3′) NO tution 1 2 12 1 2 1 2 GA TAATTT 102 I68T 0.27 0.31 0.17 0.29 0.27 0.31 0.21 0.27 104GGCCCT TCGTCT TA GA AGACTT 103 D59G 1.12 1.06 0.98 1 0.95 1.28 1.07 1.01105 TGTCCA TAAGAC GA GA GACTTT 104 N/A (D59G) 1.24 1.08 1.15 1.13 1.281.24 1.07 1.34 106 GTCCAT AAGACG AA GA ATTGTC 110 N/A 0.53 0.44 0.460.54 0.49 0.35 0.39 0.5 112 TTGATC AATTCT GG GA ATTCTG ill S17P 1.221.31 1.17 1.26 1.23 1.19 1.21 1.13 113 GAGGAA ATAACT AG GA AACATA 113M39T 58.3 ND 50.19 ND 29.1 ND 16.55 ND 115 GCAAAT CTTGAT TT GA GTAGAA114 I132T 54.78 ND 65.37 ND 33.93 ND 19.8 ND 116 TTTTTTC TTCTAG G GAACTACA 115 Q107R 56.95 57.99 42.71 48.24 25.63 27.9 13.13 12.8 117AGTCAA AAATGA AG GA TATATT 116 Y186C 37.26 37.2 30.38 30.76 20.85 20.449.23 11.01 118 GGTCTT CCACGG TC GA CAAAGA 117 D177G 54.92 55.55 14.0642.4 25.96 25.9 10.84 12.43 119 CCTTCT CCAGAC CG GA GGTCTT 118 V182A1.79 2.04 1.41 1.59 1.21 0.89 0.73 0.76 120 CCACGG TCTGGA GA GA TTGTTT119 Y186H 57.04 65.91 45.78 45.61 30.15 33.83 15.68 13.37 121 ATATTGGTCTTC CA GA CTTTTA 120 I196T 40.76 37.69 37.03 34.27 21.08 18.83 11.257.49 122 TTTGAC TATGCT GT GA AAAGTC 121 F167L 12.51 13.68 8.79 12.05 8.15.76 3.63 2.83 123 TGGATA TAGAGA GT GA GTTGGT 122 L189P 4.43 4.46 4.134.34 2.5 2.4 1.2 0.71 124 TTAATT GTTTAT AT GA TGATGG 123 N/A 23.46 26.0320.98 24.59 13.15 15.36 6.44 5.84 125 TAAGAC ACTTTG GT GA GGAGTA 124 N/A30.93 32.19 18.11 28.57 15.14 14.56 7.37 5.22 126 GTTCTT GGTGCT CT GAAACATG 125 H239R 43.8 45.03 35.09 34.98 23.07 24.46 12.12 12.5 127ATGGTA AGACAC TT GA TGAAGA 126 F228P, 57.41 74.82 54.67 60.9 34.35 48.5522.73 17.55 128 AAGGGA L229P GTAGTT CT GA AGTTCT 127 N/A 0.19 0.07 0.070.13 0.05 0.11 0.1 0.08 129 TGGTGC TCTTGG CT GA ATGATG 128 D240G 22.1622.73 18.57 20.93 10.79 10.52 5.93 5.09 130 GTAAGA CACTTT GG GA GAAGAT129 L216P, 73.63 82.24 65.4 74.19 42.42 47.99 25.24 27.14 131 AGAGAAS217P ATTTCT GT GA GGAAGA 130 S217P 76.43 88.77 69.65 70.55 49.49 46.3222.36 23.83 132 TAGAGA AATTTC TG GA TATTTC 131 I234T 33.9 31.49 26.9530.48 14.09 21.88 12.1 11.31 133 ATTCAA CTGAAG AA GA ATTTCA 132 N/A41.07 47.04 38.09 38.78 24.71 21 15.48 15.68 134 TTCAAC TGAAGA AA GAGTCTAC 133 Y273C 34.5 36.73 31.33 31.88 19.77 19.85 12.12 10.61 135TGTGAT GTTATA TC GA TAAATG 134 I249T 31.45 31.46 22.08 22.42 12.96 13.186.23 5 136 GTGGTA CATTCA GC GA TATCAG 135 N/A 39.23 45.5 35.54 35.2420.93 15.07 10.3 9.13 137 GTAAAA CCTGTC TA GA TGTACC 136 T247A 38.6538.24 29.16 29.42 19.7 17.09 10.32 8.59 138 ACCATT TATAAC AG GA TTCACC137 N/A 13 11.96 10.47 9.45 6.86 4.95 3.51 3.78 139 TCTGTT ATAAAT GG GAAACAGA 138 R252G 30.27 41.96 32.49 33.65 18.78 17.9 9.48 7.7 140 GGTGAACATACA AG GA TTGAGA 139 S267P 0.4 0.41 0.37 0.51 0.4 0.67 0.44 0.45 141GTTGCT GGGTCT GA GA TTAATT 141 I285V 12.33 10.68 9.23 8.81 5.47 4.633.05 2.94 143 CAACAT CGAATA GA GA TTTGGG 142 R308G 15.05 15.17 10.8212.69 7.52 8.04 4.16 4.56 144 AGGCTT GATGGT AA GA CATTAT 143 N/A 66.3262.98 55.96 58.5 37.77 37.26 20.55 17.46 145 ATTCAG (intron) GTAGTC CAGA TTGGGA 144 R308G 22.26 23.4 16.22 18.81 9.91 10.61 4.57 4.62 146GGCTTG ATGGTA AG GA TTTTGG 145 R308G 3.99 3.67 3.46 2.72 1.96 2.41 1.031.2 147 GAGGCT TGATGG TA GA ACAAAA 146 N294G 25.08 21.83 22.57 21.3312.96 16.13 8.62 7.59 148 CTTCAA TGAAAC GT GA TATGGT 147 Y304C 0.47 0.430.42 0.48 0.52 0.47 0.45 0.47 149 TTTGGG AGGCTT GA GA ACTACA 148 Y302C,51.08 51.83 40.09 43.73 23.76 22.55 10.14 9.52 150 AATATG K303G GTTTTGGG GA GAGAAC 149 N301G 45.3 44.63 31.68 35.7 22.72 20.85 10.83 12.4 151TACAAA TATGGT TT GA AGGACA 150 H391R 15.32 14.78 9.37 11.5 6.33 6.894.95 4.38 152 CTTCAA CTGTCC AG GA CTTTTC 101 N/A 22.82 23.57 18.25 16.178.41 7.17 4.79 ND 153 AGGAGA (intron) ATTTTG GT GA ACGTGG 242 E300G 4.855.62 3.78 4 2.98 2.75 2.38 1.96 276 GAGAAC TACAAA TA GA CGATGT 243 N/A2.36 2.43 1.69 1.87 1.53 1.72 1.16 1.17 277 TGAATT AATGTC CA GA CACAAA244 Q293R 4.09 4.48 2.43 2.81 2.2 2.01 1.68 1.74 278 ACTTCA ATGAAA CG GATACGAA 245 1333V 22.69 29.48 19.35 21.19 12.41 15.31 7.23 7.37 279TTGAGT TGGAAG AC GA CTATGG 246 S322P 22.78 30.95 22.27 19.12 16.01 16.2911.29 5.95 284 AGTATA TCTTCT CT

The gRNAs GA066/GA095, GA096, G097, GA346, matching the5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA066) protospacer sequencebut with more extensive chemical modifications of various kinds(Table 1) were synthesized. Similarly, three gRNAs (GA098, GA099, GA100)were synthesized matching the 5′-AAGATACCTGAATAACCCTC-3′ (SEQ ID NO: 15)(GA091) human protospacer sequence, as well as four gRNAs (GA067/GA101,GA102, GA103, GA347) matching the 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO:14) (GA067) cynomolgus protospacer sequence, but with more extensivechemical modifications of various kinds (Table 1). GA066 and GA095contain the same chemical composition and modification pattern, as doGA067 and GA101 with one another. It is contemplated that such gRNAs mayimprove stability against nucleases and gRNA-base editor complex, andreduce or suppress gRNA-triggered immune reaction. Each of the gRNAsdescribed in Table 8, with an equivalent amount of in vitro transcribedABE8.8 mRNA MA002 (1:1 ratio by weight), were co-transfected intoprimary human hepatocytes and primary cynomolgus hepatocytes viaMessengerMax reagent, using various dilutions to assess for editingactivity at different concentrations of test article. Three days aftertransfection, genomic DNA was harvested from the hepatocytes, and thenassessed for base editing of the target splice site with next-generationsequencing. In both human and cynomolgus hepatocytes, as high as 60%-70%editing of the target splice site (PCSK9 intron 1 splice donor; ANGPTL3intron 6 splice donor) were observed (Table 8).

TABLE 8 Chemically modified gRNAs have high editing efficiency in humanand cynomolgus primary hepatocytes Human Primary Hepatocytes- CynomolgusPrimary Hepatocytes- splice site editing % splice site editing % 2500500 100 20 2500 500 100 20 gRNA Species ng/mL ng/mL ng/mL ng/mL ng/mLng/mL ng/mL ng/mL GA066 Human/ 67.7 55.1 38.9 13.3 48.2 55.3 43.8 19.0Cyno GA096 Human/ 62.8 49.9 41.2 15.6 65.9 51.4 48.0 19.6 Cyno GA097Human/ 67.3 68.1 54.1 22.6 62.2 58.1 56.8 28.0 Cyno GA098 Human 70.751.3 36.3 10.9 ND ND ND ND GA099 Human 70.3 47.0 34.4 11.5 ND ND ND NDGA100 Human 69.2 48.6 38.5 12.4 ND ND ND ND GA066/ Cyno ND ND ND ND 61.449.0 45.9 16.0 GA101 GA102 Cyno ND ND ND ND 62.5 51.7 58.1 25.9 GA103Cyno ND ND ND ND 62.7 44.2 44.6 17.2

The method by which classical CRISPR/Cas9 disrupts a gene by ultimatelyintroducing an indel, is distinctly and significantly different thanbase editing. Specifically, base editing is used to introduce a basemutation(s) within a target window closer to the 5′ region of theprotospacer, as opposed to 3-4 bp away from the PAM, as routinely seenwith CRISPR/Cas9. Further, a target region that is highly amenable toCRISPR/Cas9 editing does not necessarily mean base editing at thatlocation will occur, and vice versa. LNPs containing either: (1) Cas9mRNA MS010 and a gRNA matching 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO:13) (GA097) protospacer sequence; or (2) ABE8.8 mRNA MA004 and gRNAmatching 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) (GA097) protospacersequence, were transfected in human primary hepatocytes. Sangersequencing of the edited genomic DNA was performed and illustrated thedifference in gene editing that occurred from CRISPR/Cas9 compared tobase editing (FIG. 11 ). The arrow highlights the main base editingposition, while the scissors highlight the general region where Cas9cuts.

To demonstrate that base editing of a splice site disrupts splicing,reverse transcription-PCR of mRNA from treated primary humanhepatocytes, using primers in exon 1 and exon 2, was performed. Resultsconfirmed that splice site disruption resulted in the use of alternativesplice donor sites within intron 1, well downstream of the in-frame TAGstop codon (FIG. 12 , Table 9). The table reports the number of mappedreads for each splice site donor as determined from next generationsequencing.

TABLE 9 Alternative splice donor sites within PCSK9 intron 1 resultingfrom editing of PCSK9 exon 1 splice-donor adenine base in primary humanhepatocytes. Number of aligned reads corresponding to: Intron IntronIntron Intron Intron Intron length length length length length lengthMapped 0 83 87 135 153 154 Sample* reads (GT donor) (CC donor) (GTdonor) (CG donor) (GC donor) (CA donor) control 7827 3187 0 0 0 0 0 (1)control 10584 5956 0 0 0 0 0 (2) control 8575 2307 0 0 0 0 0 (3) control6721 5153 0 0 0 0 0 (4) treated 8278 2368 0 243 184 984 0 (1) treated4127 1851 19 235 0 785 0 (2) treated 5694 1096 21 563 0 392 130 (3)treated 4214 2036 0 70 0 1203 0 (4) *Four different primer pairs wereused for RT-PCR of the contro /treated samples.

Example 4. PCSK9 and ANGPTL3 Off-Target Validation In Vitro

With a view towards establishing the safety of a base-editing therapyknocking down PCSK9 in the human liver in vivo, off-target mutagenesisanalysis was assessed. A list of candidate sites in the human genome foroff-target mutagenesis was assembled using two different methods. Thefirst method used bioinformatic analysis of the human genome,identifying all sites with a PAM sequence compatible with Streptococcuspyogenes Cas9 (and therefore ABE 8.8) and a protospacer sequence with upto 4 single-nucleotide mismatches with the GA066 spacer sequence5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13). The second method togenerate candidate sites used an in vitro biochemical assay, ONE-seq,that determined the propensity of a ribonucleoprotein comprising theABE8.8 base editor protein and PCSK9 gRNA(5′-CCCGCACCTTGGCGCAGCGG-3′(SEQ ID NO: 13)) to cleave oligonucleotidesin a library. The reference human genome (GRCh38) was searched for siteswith up to 6 mismatches to the protospacer sequence specified by thePCSK9 gRNA (5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13)), and sites withup to 4 mismatches plus up to 2 DNA or RNA bulges using Cas-Designerwere identified. More specifics on ONE-seq library preparation,experimental protocol, and bioinformatic analysis are described in theadditional detailed methods section.

Any cleaved oligonucleotides are PCR-amplified and undergonext-generation sequencing. Oligonucleotides with higher sequence countsreflect a higher propensity for Cas9/gRNA cleavage in vitro andrepresent the sites most likely to suffer off-target mutagenesis incells. The top site identified by the ONE-seq assay was the on-targetPCSK9 site. The list of candidate off-target sites comprises more than250 sites, and warranted further investigation (Table 10).

Primary human hepatocytes were treated with lipid nanoparticle (LNP)encapasulating ABE8.8 mRNA MA004 and PCSK9 gRNA (GA097 or GA346) at 1:1weight ratio (see Lipid Nanoparticle Formulation and Analysis fordetails of LNP preparation) Upon next-generation sequencing using theAgilent SureSelect technology (Table 11), when the observed base editingrates in control cells were subtracted from the observed base editingrates in LNP-treated cells across the on-target site and candidateoff-target sites (to account for background sequencing errors inherentin next-generation sequencing), appreciable base editing was observed atthe on-target PCSK9 target site. These results were replicated in threedifferent primary hepatocyte lots (STL, HLY, and JLP) from two male andone female patients.

Additionally, using hepatocytes from four individual donors (includinglot TLY from a female patient, in addition to the three lots listedabove), more than 50 off-target sites were assessed by performingnext-generation sequencing of targeted PCR amplicons from LNP-treatedversus untreated hepatocytes. Editing at none of these potentialoff-target sites, and only on-target editing at the PCSK9 target site,was observed (FIG. 13 ).

TABLE 10 PCSK9 gRNA (Protospacer 5′-CCCGCACCTTGGCGCAGCGG-3′(SEQ IDNO: 13)) candidate off-target sites as determined by ONE-seq Chromo-Potential Off-target sequence SEQ some Location (5′-3′) ID NO: Alignment1 55040029- CCCGCACCTTGGCGCAGCGGTGG 80 X00 55040051 1 6888039-CTAGCACC-TGCCCCAGCGGTGG 453 RNA41 6888061 1 10640222-TCCCTACCCTGGCACAGCAGGGG 454 X60 10640244 1 15101074-CCCACACTCAGACGCAGCGAGGG 455 X60 15101096 1 20712184-CCCGCAC--TGGAGCAGCAGGGA 456 RNA32 20712206 1 22256342-CTTGCACCTTGGTAGCAGCTGGGG 457 DNA41 22256365 1 25103342-CTGGCACCATGGCCCAGCAGTGG 458 X50 25103364 1 29882027-ACTGCATCTTAGTGCAGAGGTGG 459 X60 29882049 1 40866872-TCCACACCTTGGCGCACTGGAAG 460 X50 40866894 1 43692630-CATTCACCTTGTCACAGCTGGGG 461 X60 43692652 1 44163492-ACCGCACCATTGCACAGCCTAGG 462 X60 44163514 1 54256943-GCCGCACCATGGGCACAGCGGGGA 463 DNA41 54256966 1 60335313-CCCATACC-TGGCACAGCGGTGG 464 RNA31 60335335 1 64198527-CAAGCACC-TGGCTCAGAGGTGG 465 RNA41 64198549 1 64266524-ACCGCACC-TGGCACAGTGGAAG 466 RNA41 64266546 1 110096163-CCCCACCCTTGGCACAGAGCAGG 467 X60 110096185 1 121262583-CCAGCACC-TAGCGCAGAGCTGG 468 RNA41 121262605 1 145017699-CCAGCACC-TAGCGCAGAGCTGG 468 RNA41 145017721 1 155615561-CCCGCACC-TACCTCAGCAGGGG 469 RNA41 155615583 1 155751756-CCCGCACC-TACCTCAGCAGGGG 469 RNA41 155751778 1 157194781-CCCGCACCCCACCGCAGCGGGGG 470 X40 157194803 1 163263975-TCTTTACCTTGGAGCAGCTGAGG 471 X60 163263997 1 175575740-CCCGCACCACAGTGCACCGGGGG 472 X50 175575762 1 204314754-CCTCCACAATGGCGCAGGGGCGG 473 X50 204314776 1 206281058-CCAGCACC-TAGCGCAGAGCTGG 468 RNA41 206281080 1 208574946-CCAGCACC-TGGCACAGAAGAGG 474 RNA41 208574968 1 228337504-ACCGCACCAAGGTGCATAGGAGG 475 X60 228337526 1 240808285-CCAGCA-CTTGCCCCAGCAGAGG 476 RNA41 240808307 2 9138986-9139008CTCCCACCCTGGCCCAGCGGGGG 477 X40 2 11670027- ACAGCACCTTGGCCCAGAGGCGA 478X50 11670049 2 15561025- ACGGCACCTTGGGCTAGCGGGGG 479 X50 15561047 242969964- CCCGCACCCAGCTGCAGCCGAGG 480 X50 42969986 2 69112187-CCAGCACC-TGGTGCAGAGTAGG 481 RNA41 69112209 2 71667395-CCCACACCTGGGCCCAGTGGAGG 482 X40 71667417 2 72985325-CCAGCACC-TGGTGCAGCTTAGG 483 RNA41 72985347 2 85317392-TCCACACAATGGCACAGCAGAGG 484 X60 85317414 2 90356123-CCCGCACC-TGGCTCAGAGGGTC 485 RNA41 90356145 2 91447288-CCCGCACC-TGGCTCAGAGGGTC 485 RNA41 91447310 2 107914896-CGGGCACCATGGCAGCAGCGGAGG 486 DNA31 107914919 2 110633933-CGGGCACCATGGCAGCAGCGGAGG 486 DNA31 110633956 2 120922089-CCCACACCATGGCCCAGTGAGAG 487 X60 120922111 2 127571237-CCAGCACC-TGGCACAGTGAAGG 488 RNA41 127571259 2 132524147-CCCACACATGGCCACAGCAGTGG 489 X60 132524169 2 179801496-CTGGCACCTTAGCACAGAGGAGG 490 X50 179801518 2 182608809-CCCGCACCAGAGCGCAGGGGGAG 491 X50 182608831 2 197360047-GCCACACC-TGGCCCAGCAGGGG 492 RNA41 197360069 2 203642814-CCCGCACCAGGGCGCAGATGGAG 493 X50 203642836 2 203866756-TCCACACCTCAGCACAGCAGAGG 494 X60 203866778 2 219451287-ACCGCACCTTGGCCCAGTGCTGG 495 X40 219451309 2 231686140-ACCGCAGC-TGGCGCAGCATGGG 496 RNA41 231686162 3 3719723-3719745CCCGCACC-TGGCTCAGAGGGTC 485 RNA41 3 10287991- CCCGCA-TTTGCCTCAGCGGTGG497 RNA31 10288013 3 13178217- CCAGCA-CTTGGCACAGCAGTGG 498 RNA3113178239 3 14242673- CCCACACCTTGGTGTCAGCGGAGG 499 DNA21 14242696 314819436- CCTGCACC-CGGAGCAGCAGGGG 500 RNA41 14819458 3 38369509-CCTCCACCTTGGCCCAGTGGAGG 501 X40 38369531 3 52005791-CTCCCACCCTGGCGCAGAGGAGG 502 X40 52005813 3 54016522-TCAGCACC-TGGTGCAGAGGAGG 503 RNA41 54016544 3 118171416-CGAGCACCATGGCGCAGCCCCGG 504 X50 118171438 3 118171480-CCCGCA-CTTGGAGCAGCCGGCG 505 RNA31 118171502 3 139288546-GGAGCACCTTGACCCAGCAGAGG 506 X60 139288568 3 139677330-CCAGCA-CTTAGCCCAGCAGCGG 507 RNA41 139677352 3 139731008-CCCGCACCTCGGCACAGCTAGGG 508 X40 139731030 3 150408362-GCCCCATCTTGGCCCAGCGGAGG 509 X40 150408384 3 182806878-CTAGAACCATGGTGCAGAGGGGG 510 X60 182806900 4 1221653-1221675ATCCCACCTCGGCACAGCTGGGG 511 X60 4 1308120-1308142CCCGCACCGTGGTACAGCCTGTG 512 X60 4 6008382-6008404CCCGCACC-AGGCCCAGCTGGCG 513 RNA41 4 6747615-6747637CCCACACCTTGGTGCAGCTCTGT 514 X50 4 7054192-7054214ACCACACC-TGTCCCAGCGGAGG 515 RNA41 4 37229056- CACACTACTTGGCACAGAGGAGG516 X60 37229078 4 84244376- ACCGCA-CTCAGAGCAGCGGAGG 517 RNA41 842443984 114598629- TCCGCA-CTTGGCTCAGCGGGGC 518 RNA31 114598651 4 115179695-CCCCGCACTCGGAGCAGCGGCGG 519 X60 115179717 4 139511785-TCCGCACCCTGAGAGCAGTGGAGG 520 DNA41 139511808 4 141615591-CCCTGCACTTGGTGCAGCTGGGG 521 X60 141615613 4 158623208-ACCGCACC-TGACCCAGTGGTGG 522 RNA41 158623230 4 182110224-CTCCTACCATGGCACAGCTGAGG 523 X60 182110246 5 767330-767352CCCACACCAGGGCGCAGCTGAAG 524 X50 5 1516550-1516572CCAGTACCTTGGCCCAGCTTTGG 525 X50 5 77203454- ACAGCACC-TGGCACAGTGGTGG 526RNA41 77203476 5 126364896- CCCACACC-TGGAGCACCAGGGG 527 RNA41 1263649185 126628420- CAAGCACCTTGGCAGCAGCTGAGG 528 DNA31 126628443 5 173667321-CCCATGCCTTAGCACAGCGATGG 529 X60 173667343 5 178526685-CTCTATCCTTGGCCCAGCAGTGG 530 X60 178526707 6 3751269-3751291CCCGCACCTTGCCGCAGCGGCCC 531 X30 6 15092621- ATGGCACCTTGGCACATCAGAGG 532X60 15092643 6 16346963- CCCCGCACTCGGAGCAGCGGCGG 519 X60 16346985 629016770- TCCCCACACTGTCGCAGAGGAGG 533 X60 29016792 6 36310604-CCAGCACCATGGCACAGAGAGGT 534 X60 36310626 6 44047493-ACAGCACC-TGGCACAGAGGAGG 535 RNA41 44047515 6 47477658-ACCGCACCTGGCAGCAGCCGTGG 536 X50 47477680 6 73315113-CCAGCACC-TGGAGCAGCCGAGG 537 RNA31 73315135 6 138138382-CCAGCACC-TGGCACAAAGGAGG 538 RNA41 138138404 6 139537177-GAGGCACCTTGGACCAGCAGCGG 539 X60 139537199 6 148698322-CCCGCA-CTTGGAGCAGCCGGTG 540 RNA31 148698344 6 153756217-CCAGCA-CTTAGTGCAGCGCAGG 541 RNA41 153756239 6 165843913-CCCACACC-TGGAGCAGCAGGAG 542 RNA41 165843935 7 93102-93124CCCACACC-AGGCACGGCGGGGG 543 RNA41 7 5360719-5360741CCCTCACC-AGCCGCAGCAGGGG 544 RNA41 7 24849083- CACCCGACTTGGCGACAGCGGGGG545 DNA41 24849106 7 74043103- ACCGCACC-TGGCACAGAAGGGG 546 RNA4174043125 7 84150566- CCCGCACC-TGGCTCAGAGGGTC 485 RNA41 84150588 796041009- ACCACACC-TGGCCCAGCAGTGG 547 RNA41 96041031 7 98765214-CCCTCACC-GGGCACAGAGGAGG 548 RNA41 98765236 7 106882973-CCAGTACC-TGGCCCAGAGGAGG 549 RNA41 106882995 7 123763164-CCCGCACC-AGGCGCAGGTGGAG 550 RNA41 123763186 7 129225099-CAGGCACCTTGGCGGCAGCCGCGG 551 DNA31 129225122 7 139596895-TGTGCACCATGGCACAGCGGGGA 552 X60 139596917 7 147737806-CCCGCACC-TGGCTCAGAGGGTC 485 RNA41 147737828 7 150737589-CCCGCACACAGACGCAGAGCAGG 553 X60 150737611 7 153256176-TCCACACC-TGGCCCAGCTGTGG 554 RNA41 153256198 7 155527865-CCTGCAC--TGGCCCAGCAGCGG 555 RNA32 155527887 8 2485153-2485175CCTGCA-CTAGCCGCAACGGGGG 556 RNA41 8 26191893- TGAGCACCTTGGGGCAGCTGGGG557 X50 26191915 8 100957388- CCCGCA-CTTGGCGCAGCTGGCC 558 RNA31100957410 8 139813559- CCAGCACCTTGGCAGCAGCACCGG 559 DNA31 139813582 8142499068- TCCCCCACTTGGCACAGCTGGGG 560 X60 142499090 8 143383198-CCAGCA-CAGGGCGCAGAGGTGG 561 RNA41 143383220 9 33873295-CCCACCCCTTAGCACAGCAATGG 562 X60 33873317 9 34958979-CCCACACCGTGGCCCAGAGGTGG 563 X40 34959001 9 109123571-CCAGCACC-TGGCACAGCATAGG 564 RNA41 109123593 9 137012994-ACCACTACCTGGCCCAGCGGTGG 565 X60 137013016 9 137167999-TCCGCACCTTGGTCCAGCAGGGG 566 X40 137168021 9 137477162-TACGCACC-TGGCTCAGCAGAGG 567 RNA41 137477184 10 1572578-1572600AGCCCAGCATGGCGCAGAGGTGG 568 X60 10 5824416-5824438CCAGCACC-TGGCACAGAGGAGA 569 RNA41 10 16285410- ACCGCACC-TGGCCCAGATGGGG570 RNA41 16285432 10 20297215- CCCGCACC-TGGCTCAGAGGGGT 571 RNA3120297237 10 21126683- ACCGCACC-TGGCCCAGTGGAGG 572 RNA31 21126705 1028839554- CCCACATCCTGGCCCAGCAGGGG 573 X50 28839576 10 47223533-CCTGCACC-AGGCTCAGTGGGGG 574 RNA41 47223555 10 59089166-CGAGCACCTTGGCAGCAGCTGAGG 575 DNA31 59089189 10 60958888-ACCGCACC-TGGCCCAGAGTAGG 576 RNA41 60958910 10 88036335-ACAGCACC-TGGCGCAGAGCGGG 577 RNA41 88036357 10 97031490-ACTCCACCATGGCACAGCGTGGG 578 X60 97031512 10 111259337-ACCACACC-TGGCCCAGAGGAGG 579 RNA41 111259359 10 122572126-GCGGCACCTTGGCCCAGCGTGGG 580 X40 122572148 10 130560773-CCTGCACCTGAGCACAGAGGAGG 581 X50 130560795 11 2916587-2916609CCCGCACCCT-GTGCAGCCGAGG 582 RNA31 11 10630862- CCCAC-TCTTGGCACAGGGGAGG583 RNA41 10630884 11 18729719- CCCGCACC-AGGTGCAGGAGTGG 584 RNA4118729741 11 47350413- ACCGCACC-TGGCCCAGCAAAGG 585 RNA41 47350435 1147941203- GCTGCACCTTAGCACAGTGAAGG 586 X60 47941225 11 57761981-TCAGCACCTTGGCGAAGCCTTGG 587 X50 57762003 11 72870610-ACCGCACC-TGGCCCAAAGGGGG 588 RNA41 72870632 11 76659109-CCCTCACCCTGGCACAGCCGGGG 589 X40 76659131 11 117692097-TTCACACCTTGGTGCAGAGGTGA 590 X60 117692119 11 121325368-CCAGCACC-TAGCGCAGCGGCTG 591 RNA31 121325390 11 129981605-ACCGCACC-TGGCCCATCAGGGG 592 RNA41 129981627 12 3851459-3851481CCCGCACC-TGGCTCAGCGGGTC 593 RNA31 12 32850805- TCAGCACC-TGGAGCAGCCGAGG594 RNA41 32850827 12 48000120- CCCCACCCTTGGTGCAGAGGAGG 595 X50 4800014212 53328526- CATACACCTTGCCGCAGCCAGGG 596 X60 53328548 12 54190321-CACACACCTTAGCACAGCCAAGG 597 X60 54190343 12 65971780-CTAGCACC-TGGCACAGTGGTGG 598 RNA41 65971802 12 95590363-ACCGTACC-TGGCCCAGAGGAGG 599 RNA41 95590385 12 106690762-GCCCCACCTTAATGCAGCGGGGG 600 X50 106690784 12 114491014-CCAGCTACATGGCCCAGAGGAGG 601 X60 114491036 12 127431260-CCCACACCAT-GCGCAGAGTGGG 602 RNA41 127431282 13 25788073-CCCGCACCCAGGC-CAGCTGTGG 603 RNA31 25788095 13 69424400-CCAGCACC-TGCTGCAGCGATGG 604 RNA41 69424422 13 85917572-CCCGCACC-TGGCTCAGAGGGTC 485 RNA41 85917594 13 98388403-TCCGCACCTTGGCCCAGTTGAAG 605 X50 98388425 14 20813343-ACAGCACCTTGGCAGCAGAGGTGG 606 DNA31 20813366 14 24408232-CCCGCACC-AGGCTCAGCAGCAG 607 RNA41 24408254 14 38663021-CCAGCACC-TGGCACAGCCAGGG 608 RNA41 38663043 14 65250553-GCTGCACCCTGCCACAGAGGAGG 609 X60 65250575 14 89310097-CCAGCACCCTAGTGCAGAGTGGG 610 X60 89310119 14 90554536-CCCGCACC-TGGCACAGCACCAG 611 RNA41 90554558 14 100213915-CCCG-ACCCTGACGCAGCCTGGG 612 RNA41 100213937 14 101153383-CCCGCACCCTATCACAGCAGAGG 613 X50 101153405 14 102929418-CTCGCACC-AGGCGCAGCCGTGG 614 RNA31 102929440 15 42252041-CCCGCACC-TGGCTCAGCGGGTC 593 RNA31 42252063 15 47827093-CATGTACCTTGCCACAGCAGGGG 615 X60 47827115 15 69452825-CGAGCACCTTGGCGGCAGCTGAGG 616 DNA31 69452848 15 86059010-TCCACACCTAGGCACAGCCTAGG 617 X60 86059032 15 94089230-CCAGCACCAAGGCTCAGCTGAGG 618 X50 94089252 15 99993270-ACCGCACC-TGGCACATTGGAGG 619 RNA41 99993292 16 1003178-1003200CCCACACCTTGGCCCAGCCCTGG 620 X40 16 2092489-2092511GCCGCACC-TGCCGCAGCCGTGG 621 RNA31 16 3089598-3089620CCCGCA-CTGGGTGCAGTGGTGG 622 RNA31 16 14882693- CCCACACCCAGGTGCAGCGGCAG623 X50 14882715 16 16097875- CCAGCACC-TGGCACAGCAGGTG 624 RNA41 1609789716 16281338- CCCACACCCAGGTGCAGCGGCAG 623 X50 16281360 16 18430777-CCCACACCCAGGTGCAGCGGCAG 623 X50 18430799 16 18513312-CCCACACCCAGGTGCAGCGGCAG 623 X50 18513334 16 21358246-CCCGCATCTTGGTGCAGCTGCTT 625 X50 21358268 16 28383449-ACCGCACCATGGTGCAGCTGGGC 626 X50 28383471 16 28731839-ACCGCACCATGGTGCAGCTGGGC 626 X50 28731861 16 29792483-CCAACACC-TGGCGCAGAGTAGG 627 RNA41 29792505 16 29863301-CTCGTACCAAAGCGCAGAGGAGG 628 X60 29863323 16 30783356-CCCGCA-CTCGGTGCAGCGGTAT 629 RNA41 30783378 16 31081352-CCCGCA-CTGGGTGCAGCGGAAG 630 RNA31 31081374 16 31436901-GCCGCA-CTTGGCGCAGCTGTGG 631 RNA21 31436923 16 67748649-CTAACACCTTGGTGCAGAGGTGG 632 X50 67748671 16 78768901-CCTACACCATGGCGCAGCGTCAG 633 X50 78768923 16 81278358-CCAGCACC-TGGCACATCGGTGG 634 RNA31 81278380 16 88044071-CCCGCATCTTGGTGCAGCTGCTT 625 X50 88044093 16 88839686-CCCGCA-CATGGTGCAGTGGCTG 635 RNA41 88839708 17 7223899-7223921CCCAC-CCCTGGCCCAGCCGAGG 636 RNA41 17 7462659-7462681TGCACACCTTGGCGCAGTGGGGG 637 X40 17 7511796-7511818CCAGCACC-TGGCACAGGGGAGG 638 RNA31 17 19391599- CCCACACC-AGGCACAGAGGAGG639 RNA41 19391621 17 26736056- CCCGCACC-TGGCTCAGAGGGTC 485 RNA4126736078 17 28662198- GTAGCACGTTGACGCAGCAGCGG 640 X60 28662220 1738870195- GCCGCACC-GGGCGCAGTTGGGG 641 RNA41 38870217 17 45753295-CCCACACC-TGCCCCAGAGGTGG 642 RNA41 45753317 17 45993823-CCCACAGC-TGGCCCAGCAGGGG 643 RNA41 45993845 17 63929863-TCAGCACCTTGGCACAGCTGAAG 644 X50 63929885 17 79154410-ACAGCACCTCGGTGCAGCAGAGA 645 X60 79154432 17 81131740-CCCTC-CCATGGCGCAGCTTCGG 646 RNA41 81131762 18 3448410-3448432CCCGCACC-GGGCGCAGCAGCTG 647 RNA31 18 39380982- CATGCTACATGGCGCAGCGGTAG648 X60 39381004 18 58247634- CTCGCACCCGAACGCAGCAGAGG 649 X60 5824765618 59917417- TCCCCACCCCAGCGCAGCAGAGG 650 X60 59917439 18 78993257-CGAAGACCTTGGCGCAGAAGCGG 651 X60 78993279 19 1104611-1104633CCCGCACCTTGGCCTAGCGCGGT 652 X40 19 1274125-1274147TCTGCACCTTGGCGCAGCTGGAG 653 X40 19 2209581-2209603CTCCCACCTTAAAGCAGCGGTGG 654 X50 19 5543277-5543299ACCGCACC-TGGCCCAGAGTGGG 655 RNA41 19 6431799-6431821CCCCCACCTTGGCCCAGCGTTGG 656 X30 19 10872441- GCCACACC-TCGCACAGCGGTGG 657RNA41 10872463 19 11391283- ACCACACCTTGCTGCAGAGGAGG 658 X50 11391305 1911406443- CCCGCA-GAGGGCGCAGCGGCTG 659 RNA41 11406465 19 12747607-CCAGCAC-TGGCTCAGCAGAGG 660 RNA32 12747629 19 36110180-ACCGCACC-TGGCCCAAAGGGGG 588 RNA41 36110202 19 46709648-ACCGCACC-CGGCCCAGTGGAGG 661 RNA41 46709670 19 49720095-CCTGCACTCAGGCGCAGACGGGG 662 X60 49720117 19 50490618-GCTGCACCTTGGCACAGTGGAGG 663 X40 50490640 19 51068476-CCCACACCCAGGCCCAGAGGAGG 664 X50 51068498 19 51606728-CCTGCACCT--GCACAGCAGGGG 665 RNA32 51606750 19 53775475-CGAGCACCTTGGCAGCAGCTGAGG 575 DNA31 53775498 19 56088833-CTCGCACC-TGGTGCAGCACCGG 666 RNA41 56088855 20 13900645-GCCGCAC--AGGCACAGCGGCGG 667 RNA32 13900667 20 23109029-TCAGCACCTTGGCACATCCAGGG 668 X60 23109051 20 31859435-CCTGCACCTTTGCACAGCACTGG 669 X50 31859457 20 45967752-CCCGCA-TGTGGCGCAGCAGTGT 670 RNA41 45967774 20 49558961-CCCAGCACTTGGTGAAGCGGAGG 671 X60 49558983 21 37176555-CCCACAC-TGGTGCAGAGGTGG 672 RNA32 37176577 22 22912001-CCTGCACCATGGTGCACCGGCAG 673 X50 22912023 22 25849024-CCCGCAC-AGCCGCAGAGGAGG 674 RNA32 25849046 22 27735501-ACCGCACC-TGGCACAGCTTGGG 675 RNA41 27735523 22 49971621-CCCACACCCTGGCCCAGCCTCGG 676 X50 49971643 X 1842130-CCAGCACTTTGGTACAGCATAGG 677 X60 1842152 X 23566446-CCCGCACC-TGGCTCAGAGGGGT 571 RNA31 23566468 X 50814798-CCCGCACCCTGGCTCAGTGTTGG 678 X40 50814820 X 76172984-CCCGCAC-AGGCGCAGAGGTGG 679 RNA22 76173006 X 83385071-CCAGCACC-TGGTCCAGTGGAGG 680 RNA41 83385093 X 107536875-CCAGCACC-TGGCACAGAGTAGG 681 RNA41 107536897

TABLE 11 PCSK9 gRNA GA346 off-target site validation in human primaryhepatocytes STL HLY JLP STL HLY JLP Chro- Lot #: Treat- Treat- Treat-Con- Con- Con- Average Average Net mosome Location ed ed ed trol troltrol treated untreated Editing 1  55040029- 42.18 49.76 78.54 0.13 0.190.03 56.83 0.12 56.71 55040051 1  54256943- 0 0.07 0.04 0.1 0 0.03 0.040.04 −0.01 54256966 1  22256342- 0 0 0.02 0 0.07 0.02 0.01 0.03 −0.0222256365 1  10640222- 0 0 0 0 0.04 0 0.00 0.01 −0.01 10640244 1 6888039- 0 0 0.03 0.03 0.05 0.03 0.01 0.04 −0.03 6888061 1  60335313- 00.02 0.01 0.03 0 0.02 0.01 0.02 −0.01 60335335 1  64198527- 0 0.07 00.02 0.06 0 0.02 0.03 0.00 64198549 1  64266524- 0 0.04 0.05 0.02 0 0.050.03 0.02 0.01 64266546 1 121262583- 0.02 0.04 0.05 0.02 0 0.04 0.040.02 0.02 121262605 1  20712184- 0.05 0.05 0.03 0.03 0.06 0.06 0.04 0.05−0.01 20712206 1 240808285- 0.06 0.03 0.01 0 0.06 0.02 0.03 0.03 0.01240808307 1 145017699- 0.05 0.05 0.08 0.07 0.14 0.05 0.06 0.09 −0.03145017721 1 155615561- 0 0.04 0.01 0 0.05 0.03 0.02 0.03 −0.01 1556155831 155751756- 0.02 0 0.06 0.01 0.09 0.07 0.03 0.06 −0.03 155751778 1206281058- 0 0.08 0.08 0.02 0.07 0 0.05 0.03 0.02 206281080 1 208574946-0 0 0 0 0 0.05 0.00 0.02 −0.02 208574968 1  15101074- 0 0 0.05 0.02 0.060 0.02 0.03 −0.01 15101096 1  25103342- 0.06 0 0.03 0.03 0.03 0 0.030.02 0.01 25103364 1  29882027- 0.03 0 0.04 0.05 0 0 0.02 0.02 0.0129882049 1  40866872- 0 0 0.05 0.02 0 0 0.02 0.01 0.01 40866894 1 43692630- 0 0 0 0.02 0.04 0.01 0.00 0.02 −0.02 43692652 1  44163492- 00 0 0 0 0.03 0.00 0.01 −0.01 44163514 1 110096163- 0 0 0.02 0.03 0 0.030.01 0.02 −0.01 110096185 1 157194781- 0 0 0.03 0 0 0 0.01 0.00 0.01157194803 1 163263975- 0 0 0 0 0 0 0.00 0.00 0.00 163263997 1 175575740-0 0.05 0.06 0.07 0 0 0.04 0.02 0.01 175575762 1 204314754- 0 0.08 0.040.04 0.02 0.06 0.04 0.04 0.00 204314776 1 228337504- 0.02 0 0.01 0.050.05 0.01 0.01 0.04 −0.03 228337526 2 107914896- 0.04 0 0 0.11 0 0 0.010.04 −0.02 107914919 2 110633933- 0 0 0.02 0.03 0 0.06 0.01 0.03 −0.02110633956 2  69112187- 0 0 0 0 0.07 0 0.00 0.02 −0.02 69112209 2 72985325- 0.03 0 0 0.01 0 0.05 0.01 0.02 −0.01 72985347 2  90356123-0.05 0 0 0.01 0 0 0.02 0.00 0.01 90356145 2 127571237- 0.03 0.04 0.01 00.04 0.02 0.03 0.02 0.01 127571259 2 197360047- 0 0.04 0 0 0 0.05 0.010.02 0.00 197360069 2 231686140- 0.05 0.12 0 0.02 0 0 0.06 0.01 0.05231686162 2  9138986- 96.7 99.46 96.44 97.34 99.52 96.8 97.53 97.89−0.35 9139008 2  11670027- 0.03 0 0.02 0 0 0.11 0.02 0.04 −0.02 116700492  15561025- 0 0 0 0.06 0.06 0.06 0.00 0.06 −0.06 15561047 2  42969964-0.03 0 0.04 0.05 0.08 0.03 0.02 0.05 −0.03 42969986 2  71667395- 0 0.040.02 0.05 0.08 0 0.02 0.04 −0.02 71667417 2  85317392- 0.02 0.06 0 0.010 0.03 0.03 0.01 0.01 85317414 2 120922089- 0.03 0.03 0.03 0.01 0 0.050.03 0.02 0.01 120922111 2 132524147- 0 0.05 0 0.06 0.08 0.08 0.02 0.07−0.06 132524169 2 179801496- 0.03 0.04 0 0.01 0 0.01 0.02 0.01 0.02179801518 2 182608809- 0 0 0.09 0.14 0 0 0.03 0.05 −0.02 182608831 2203866756- 0.02 0.11 0.04 0 0.04 0.01 0.06 0.02 0.04 203866778 2219451287- 0 0 0.02 0 0.08 0.02 0.01 0.03 −0.03 219451309 3  14242673-0.03 0 0 0.04 0 0.01 0.01 0.02 −0.01 14242696 3  10287991- 0 0 0 0 00.06 0.00 0.02 −0.02 10288013 3  13178217- 0.03 0 0.02 0.03 0.03 0.010.02 0.02 −0.01 13178239 3 118171480- 0 0 0 0.05 0 0 0.00 0.02 −0.02118171502 3  14819436- 0.02 0 0 0.01 0 0.03 0.01 0.01 −0.01 14819458 3 54016522- 0.06 0 0 0.01 0.05 0 0.02 0.02 0.00 54016544 3 139677330- 0 00 0.06 0 0.03 0.00 0.03 −0.03 139677352 3  38369509- 0.03 0 0 0 0 0 0.010.00 0.01 38369531 3  52005791- 0.03 0 0.04 0 0 0.02 0.02 0.01 0.0252005813 3 118171416- 0.15 0.06 0 0 0 0.03 0.07 0.01 0.06 118171438 3139288546- 0 0.02 0.04 0.03 0 0 0.02 0.01 0.01 139288568 3 139731008- 00.02 0.07 0.03 0 0 0.03 0.01 0.02 139731030 3 150408362- 0 0 0.04 0 00.04 0.01 0.01 0.00 150408384 3 182806878- 0.09 0 0.04 0 0 0.05 0.040.02 0.03 182806900 4 139511785- 0 0 0 0.03 0 0.03 0.00 0.02 −0.02139511808 4  84244376- 0.03 0 0.05 0.06 0 0.01 0.03 0.02 0.00 84244398 4114598629- 0 0 0.02 0 0 0 0.01 0.00 0.01 114598651 4  6008382- 0 0 0.040 0 0.12 0.01 0.04 −0.03 6008404 4  7054192- 0.14 0.02 0 0.02 0.07 0.010.05 0.03 0.02 7054214 4 158623208- 0 0 0.12 0 0 0 0.04 0.00 0.04158623230 4  1221653- 0.05 0 0.01 0.03 0 0.05 0.02 0.03 −0.01 1221675 4 1308120- 0.03 0 0.02 0 0 0 0.02 0.00 0.02 1308142 4  6747615- 0.03 00.05 0.02 0 0.04 0.03 0.02 0.01 6747637 4  37229056- 0.03 0.02 0 0.05 00.05 0.02 0.03 −0.02 37229078 4 115179695- 0 0 0 0 0 0 0.00 0.00 0.00115179717 4 141615591- 0 0 0.05 0 0 0 0.02 0.00 0.02 141615613 4182110224- 0.04 0 0.05 0.05 0.06 0.02 0.03 0.04 −0.01 182110246 5126628420- 0.03 0.07 0 0.05 0 0 0.03 0.02 0.02 126628443 5  77203454-0.06 0 0.07 0.1 0 0.05 0.04 0.05 −0.01 77203476 5 126364896- 0.03 0 0 00 0 0.01 0.00 0.01 126364918 5   767330- 0.09 0.04 0.05 0.03 0.06 0.040.06 0.04 0.02 767352 5  1516550- 0 0.06 0 0 0 0.03 0.02 0.01 0.011516572 5 173667321- 0.02 0 0.01 0.03 0.04 0.02 0.01 0.03 −0.02173667343 5 178526685- 0 0 0 0 0.03 0.03 0.00 0.02 −0.02 178526707 6 44047493- 0.06 0.02 0.04 0.09 0 0.05 0.04 0.05 −0.01 44047515 6 73315113- 0 0 0 0.07 0.04 0 0.00 0.04 −0.04 73315135 6 148698322- 0 00.13 0.07 0 0.11 0.04 0.06 −0.02 148698344 6 153756217- 0 0 0.01 0.070.11 0 0.00 0.06 −0.06 153756239 6 138138382- 0.06 0 0 0.04 0 0.03 0.020.02 0.00 138138404 6 165843913- 0.03 0.07 0 0.05 0 0.03 0.03 0.03 0.01165843935 6  3751269- 0 0 0 0 0 0 0.00 0.00 0.00 3751291 6  15092621- 00.03 0.02 0.01 0 0 0.02 0.00 0.01 15092643 6  16346963- 0.07 0 0 0 0 00.02 0.00 0.02 16346985 6  29016770- 0 0 0 0.02 0.08 0.02 0.00 0.04−0.04 29016792 6  36310604- 0.06 0 0.06 0.01 0.1 0.09 0.04 0.07 −0.0336310626 6  47477658- 0.2 0 0 0.07 0 0.03 0.07 0.03 0.03 47477680 6139537177- 0 0 0 0.03 0 0 0.00 0.01 −0.01 139537199 7  24849083- 0 0.050.06 0.03 0 0 0.04 0.01 0.03 24849106 7 129225099- 0 0 0 0 0 0 0.00 0.000.00 129225122 7   93102- 0.37 0 0.28 0.04 0 0.05 0.22 0.03 0.19 93124 7 5360719- 0 0.03 0.07 0.02 0 0.1 0.03 0.04 −0.01 5360741 7  74043103-0.1 0 0.07 0.04 0 0.04 0.06 0.03 0.03 74043125 7  96041009- 0.17 0.090.03 0.03 0.11 0.11 0.10 0.08 0.01 96041031 7  98765214- 0 0 0 0 0.050.03 0.00 0.03 −0.03 98765236 7 106882973- 0 0.12 0.02 0.09 0.05 0.020.05 0.05 −0.01 106882995 7 123763164- 0.08 0.29 0 0 0 0.13 0.12 0.040.08 123763186 7 147737806- 0 44.29 0.19 0.11 43.85 0 14.83 14.65 0.17147737828 7 153256176- 0 0.05 0.04 0 0 0.03 0.03 0.01 0.02 153256198 7155527865- 0 0 0 0 0 0 0.00 0.00 0.00 155527887 7 139596895- 0 0.03 0.030.03 0.03 0 0.02 0.02 0.00 139596917 7 150737589- 0.05 0.13 0.05 0.010.05 0.18 0.08 0.08 0.00 150737611 8 139813559- 0 0.06 0 0 0.06 0 0.020.02 0.00 139813582 8  2485153- 0.05 0 0.03 0.03 0 0.06 0.03 0.03 0.002485175 8 100957388- 0 0 0 0.03 0 0 0.00 0.01 −0.01 100957410 8143383198- 0.02 0.11 0.04 0.11 0.09 0 0.06 0.07 −0.01 143383220 8 26191893- 0 0.06 0 0.02 0.13 0.02 0.02 0.06 −0.04 26191915 8 142499068-0.03 0 0 0 0.08 0 0.01 0.03 −0.02 142499090 9 137012994- 0.05 0 0.070.02 0 0.03 0.04 0.02 0.02 137013016 9 137167999- 0.03 0 0 0 0 0.03 0.010.01 0.00 137168021 9 109123571- 0.03 0 0 0.02 0 0 0.01 0.01 0.00109123593 9 137477162- 0.05 0.06 0.02 0.02 0.03 0.05 0.04 0.03 0.01137477184 9  33873295- 0.05 0 0 0 0 0 0.02 0.00 0.02 33873317 9 34958979- 0.1 0.03 0 0.01 0 0.01 0.04 0.01 0.04 34959001 10  28839554-0 0.18 0.08 0.11 0 0 0.09 0.04 0.05 28839576 10  59089166- 0 0 0 0 0.060 0.00 0.02 −0.02 59089189 10  97031490- 0.1 0.12 0 0.01 0.03 0.09 0.070.04 0.03 97031512 10 122572126- 0 0.05 0.03 0 0 0 0.03 0.00 0.03122572148 10 130560773- 0.03 0.02 0.03 0.02 0 0 0.03 0.01 0.02 13056079510  5824416- 0 0.09 0.01 0 0 0 0.03 0.00 0.03 5824438 10  16285410- 0.080.09 0 0.05 0 0.03 0.06 0.03 0.03 16285432 10  20297215- 0 0 0 0 0 00.00 0.00 0.00 20297237 10  21126683- 0.08 0.09 0 0.05 0 0 0.06 0.020.04 21126705 10  47223533- 0 0 0.02 0.03 0 0.03 0.01 0.02 −0.0147223555 10  60958888- 0.1 0 0.12 0 0.14 0.03 0.07 0.06 0.02 60958910 10 88036335- 0.03 0 0.02 0.02 0 0.02 0.02 0.01 0.00 88036357 10 111259337-0.08 0 0 0.09 0 0 0.03 0.03 0.00 111259359 10  1572578- 0.03 0 0.03 00.05 0.05 0.02 0.03 −0.01 1572600 11  47941203- 0 0.05 0.02 0 0 0.030.02 0.01 0.01 47941225 11  57761981- 0 0 0.02 0.01 0 0.08 0.01 0.03−0.02 57762003 11  76659109- 0 0 0 0 0 0.01 0.00 0.00 0.00 76659131 11117692097- 0 0.04 0 0.01 0 0.02 0.01 0.01 0.00 117692119 11  10630862-0.03 0 0 0.02 0 0.06 0.01 0.03 −0.02 10630884 11  18729719- 0 0 0.060.06 0.1 0.08 0.02 0.08 −0.06 18729741 11  47350413- 0.05 0 0 0.06 0.070 0.02 0.04 −0.03 47350435 11  72870610- 0 0 0 0 0 0.24 0.00 0.08 −0.0872870632 11  2916587- 0 0 0.01 0 0 0 0.00 0.00 0.00 2916609 11121325368- 0 0.04 0.09 0.07 0.06 0.05 0.04 0.06 −0.02 121325390 11129981605- 0 0 0.04 0.06 0.13 0.04 0.01 0.08 −0.06 129981627 12 3851459- 0 0 0 0 0 0 0.00 0.00 0.00 3851481 12  32850805- 0.06 0 0.070.05 0.07 0 0.04 0.04 0.00 32850827 12  65971780- 0.06 0.04 0.03 0 0 00.04 0.00 0.04 65971802 12  95590363- 0 0.18 0 0.08 0 0.08 0.06 0.050.01 95590385 12 127431260- 0.05 0.04 0.11 0.06 0.12 0.04 0.07 0.07−0.01 127431282 12 48000120- 0 0 0.02 0 0.06 0.02 0.01 0.03 −0.0248000142 12 53328526- 0 0 0 0.06 0.09 0.02 0.00 0.06 −0.06 53328548 1254190321- 0.06 0.02 0.03 0.01 0 0.05 0.04 0.02 0.02 54190343 12106690762- 0 0 0 0.01 0 0.01 0.00 0.01 −0.01 106690784 12 114491014-0.11 0 0 0.05 0.1 0.05 0.04 0.07 −0.03 114491036 13  69424400- 0 0.040.03 0.01 0.13 0 0.02 0.05 −0.02 69424422 13  25788073- 0.05 0.04 0.040.03 0.05 0 0.04 0.03 0.02 25788095 13  98388403- 0 0.06 0.02 0 0 0 0.030.00 0.03 98388425 14  20813343- 0.05 0.02 0.01 0.02 0 0.05 0.03 0.020.00 20813366 14  24408232- 0 0.03 0.07 0.06 0 0.07 0.03 0.04 −0.0124408254 14  38663021- 0.08 0 0.01 0.06 0 0 0.03 0.02 0.01 38663043 14100213915- 0 0 0 0.03 0 0 0.00 0.01 −0.01 100213937 14  90554536- 0 00.01 0.06 0 0.03 0.00 0.03 −0.03 90554558 14 102929418- 0 0 0.03 0.03 00 0.01 0.01 0.00 102929440 14  65250553- 0.04 0 0 0.04 0 0 0.01 0.010.00 65250575 14  89310097- 0.06 0 0.06 0.03 0.06 0.04 0.04 0.04 0.0089310119 14 101153383- 0 0.04 0 0 0 0 0.01 0.00 0.01 101153405 15 69452825- 0 0 0.03 0.04 0 0.03 0.01 0.02 −0.01 69452848 15  42252041- 00 0 0 0 0 0.00 0.00 0.00 42252063 15  99993270- 0.03 0 0.02 0 0 0 0.020.00 0.02 99993292 15  47827093- 0.08 0.04 0.02 0.04 0 0 0.05 0.01 0.0347827115 15  86059010- 0.05 0.02 0.07 0.05 0.05 0.01 0.05 0.04 0.0186059032 15  94089230- 0.11 0.04 0.04 0.07 0.02 0.02 0.06 0.04 0.0394089252 16  3089598- 0 0.03 0.01 0 0.07 0 0.01 0.02 −0.01 3089620 16 30783356- 0 0 0 0.04 0 0.02 0.00 0.02 −0.02 30783378 16  31081352- 0.030 0.04 0 0 0 0.02 0.00 0.02 31081374 16  31436901- 0.02 0.02 0.01 0.010.05 0.03 0.02 0.03 −0.01 31436923 16  2092489- 0.05 0 0 0 0.03 0 0.020.01 0.01 2092511 16  16097875- 0.03 0.04 0.02 0.04 0.04 0.14 0.03 0.07−0.04 16097897 16  29792483- 0.06 0.03 0.11 0.11 0 0.03 0.07 0.05 0.0229792505 16  88839686- 0.03 0 0 0 0 0.04 0.01 0.01 0.00 88839708 16 81278358- 0 0 0 0 0 0.04 0.00 0.01 −0.01 81278380 16  1003178- 0.09 00.04 0.02 0 0 0.04 0.01 0.04 1003200 16  14882693- 0.06 0.03 0.07 0.09 00.01 0.05 0.03 0.02 14882715 16  16281338- 0.02 0.04 0.06 0.06 0 0.060.04 0.04 0.00 16281360 16  18430777- 0.08 0 0.09 0.07 0.05 0.02 0.060.05 0.01 18430799 16  18513312- 0.04 0 0 0.02 0 0.07 0.01 0.03 −0.0218513334 16  21358246- 0 0 0 0.02 0 0.02 0.00 0.01 −0.01 21358268 16 28383449- 0.02 0.1 0.03 0.03 0.04 0.01 0.05 0.03 0.02 28383471 16 28731839- 0.05 0.02 0.05 0.02 0 0.04 0.04 0.02 0.02 28731861 16 29863301- 0 0 0 0.03 0 0 0.00 0.01 −0.01 29863323 16  67748649- 0 00.03 0 0 0 0.01 0.00 0.01 67748671 16  78768901- 0.04 0 0.01 0 0.02 0.030.02 0.02 0.00 78768923 16  88044071- 0 0 0 0 0 0 0.00 0.00 0.0088044093 17  7223899- 0 0 0 0.02 0 0.06 0.00 0.03 −0.03 7223921 17 7511796- 0 0.02 0.05 0.04 0.09 0 0.02 0.04 −0.02 7511818 17  19391599-0.03 0.03 0.1 0.05 0.06 0.06 0.05 0.06 0.00 19391621 17  26736056- 0.030.08 0 0.04 0 0 0.04 0.01 0.02 26736078 17  38870195- 0 0.03 0.05 0.030.07 0 0.03 0.03 −0.01 38870217 17  45753295- 0.03 0 0.02 0.04 0 0.040.02 0.03 −0.01 45753317 17  45993823- 0 0.06 0.02 0.05 0.07 0 0.03 0.04−0.01 45993845 17  81131740- 0 0 0 0.01 0 0.03 0.00 0.01 −0.01 8113176217  7462659- 0.05 0 0.04 0.01 0.06 0 0.03 0.02 0.01 7462681 17 28662198- 0 0 0 0.07 0 0.03 0.00 0.03 −0.03 28662220 17  63929863- 0.070.02 0.03 0.01 0.02 0.03 0.04 0.02 0.02 63929885 17  79154410- 0 0 0.030.02 0 0 0.01 0.01 0.00 79154432 18  3448410- 0.03 0 0 0 0 0 0.01 0.000.01 3448432 18  39380982- 0 0.04 0.03 0.01 0 0.05 0.02 0.02 0.0039381004 18  58247634- 0.08 0.02 0 0 0.05 0.03 0.03 0.03 0.01 5824765618  59917417- 0.06 0 0 0.02 0 0 0.02 0.01 0.01 59917439 18  78993257- 00 0.04 0 0.11 0.02 0.01 0.04 −0.03 78993279 19  53775475- 0.03 0 0.020.02 0 0 0.02 0.01 0.01 53775498 19  11406443- 0.02 0 0.08 0.02 0 0.10.03 0.04 −0.01 11406465 19  10872441- 0.06 0 0 0.03 0 0.03 0.02 0.020.00 10872463 19  36110180- 0 0 0 0.13 0 0 0.00 0.04 −0.04 36110202 19 46709648- 0 0 0.09 0.03 0 0.12 0.03 0.05 −0.02 46709670 19  12747607-0.03 0 0.06 0.04 0.05 0.02 0.03 0.04 −0.01 12747629 19  56088833- 0 0.070 0.05 0.14 0 0.02 0.06 −0.04 56088855 19  51606728- 0.02 0 0.01 0.01 00.06 0.01 0.02 −0.01 51606750 19  1104611- 0 0 0 0 0 0 0.00 0.00 0.001104633 19  1274125- 0 0.05 0.02 0.05 0 0 0.02 0.02 0.01 1274147 19 2209581- 0 0 0 0.02 0.07 0 0.00 0.03 −0.03 2209603 19  6431799- 0 0 0 00 0.03 0.00 0.01 −0.01 6431821 19  11391283- 0.03 0.04 0.03 0.07 0.10.03 0.03 0.07 −0.03 11391305 19  49720095- 0.04 0 0 0 0 0.1 0.01 0.03−0.02 49720117 19  50490618- 0 0.05 0.02 0 0.03 0 0.02 0.01 0.0150490640 19  51068476- 0.07 0.06 0.1 0.08 0 0.02 0.08 0.03 0.04 5106849820  45967752- 0 0 0 0 0 0.03 0.00 0.01 −0.01 45967774 20  13900645- 0.030 0.03 0.04 0.05 0.06 0.02 0.05 −0.03 13900667 20  23109029- 0 0 0.01 00 0.01 0.00 0.00 0.00 23109051 20  31859435- 0 0.02 0 0 0 0.03 0.01 0.010.00 31859457 20  49558961- 0.04 0.09 0 0 0.07 0 0.04 0.02 0.02 4955898321  37176555- 0 0 0.03 0.07 0.05 0 0.01 0.04 −0.03 37176577 22 27735501- 0 0.08 0 0 0.04 0.05 0.03 0.03 0.00 27735523 22  25849024-0.03 0.04 0.01 0 0 0.02 0.03 0.01 0.02 25849046 22  22912001- 0 0 0.01 00.05 0 0.00 0.02 −0.01 22912023 22  49971621- 0 0.15 0.06 0 0.11 0.030.07 0.05 0.02 49971643 X  1842130- 0 0 0 0 0 0 0.00 0.00 0.00 1842152 X 50814798- 0 0 0 0.06 0 0 0.00 0.02 −0.02 50814820 X  23566446- 0 0 0 00 0 0.00 0.00 0.00 23566468 X  83385071- 0 0.08 0 0 0 0 0.03 0.00 0.0383385093 X  76172984- 0 0 0.03 0 0 0.05 0.01 0.02 −0.01 76173006 X107536875- 0 0 0 0.03 0 0 0.00 0.01 −0.01 107536897

Adenine base editors have been reported to induce gRNA-independent RNAediting via the deoxyadenosine deaminase domain. To assess forgRNA-independent RNA editing, primary human hepatocytes were treatedwith mRNA and PCSK9 gRNA (n=4 biological replicates), SpCas9 mRNA andgRNA (n=4), or were untreated (n=4). RNA was extracted after 2 days, andprocessed as described in the additional detailed methods section.Comparing the RNA profiles of the ABE8.8- and SpCas9-treated hepatocyteswith untreated hepatocytes, no substantial RNA edits in theABE8.8-treated hepatocytes was observed (FIG. 14 ). Each replicate wascompared against each of four untreated hepatocyte samples to eliminateany positions with editing that were common to both conditions. Thejitter plots portray transcriptomic loci with editing in the treatedsample (number indicates total edited loci identified in the treatedsample, boxplot indicates median ±interquartile range of proportion ofedited reads across all edited loci in the sample).

Modifications and/or truncations to either the spacer or tracr portionof the gRNA was assessed by alteration of the GA066 spacer(5′-CCCGCACCTTGGCGCAGCGG-3′(SEQ TD NO: 13)) (Table 12). Themodifications to the guide can serve to improve on-target editingefficiency and/or improve off-target editing efficiency. Each of thegRNAs with an equivalent amount of in vitro transcribed ABE8.8 mRNA (1:1ratio by weight) were co-transfected into primary human hepatocytes andprimary cynomolgus hepatocytes and processed as described in detailedmethods.

TABLE 12 Modifications and/or truncations of the PCSK9 guide GA346results in high- level editing in human primary hepatocytes HumanPrirmary Hepatocytes-Editing % at Position 6 (Dose, Replicate #) 25002500 1250 1250 625 625 312.5 312.5 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mLng/mL ng/mL gRNA rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 GA346 30.6935.58 19.99 34.67 27.13 16.93 12.56 9.92 GA376 38.15 33.98 31.43 25.2824.02 22.03 14.81 12.02 GA377 35.11 37.31 26.39 31.48 21.39 23.19 15.3414.9 GA380 38.5 35.38 32.41 32.43 25.28 20.36 12.85 ND GA381 29.7 31.224.12 29.23 17.32 18.43 11.44 11 GA382 34.56 30.02 22.5 24.45 18.4815.64 8.45 8.86 GA383 42.2 34.95 32.76 31.79 22.24 24.17 13.27 13.26GA384 8.49 8.5 7.07 6.51 5.03 4.25 2.61 2.7 GA385 40.2 29.05 26.39 21.4318.88 16.66 11.76 7.63 GA386 43.73 30.67 31.57 26.5 27.51 19.19 16.9413.41 GA387 22.61 16.64 17.25 12.78 11.55 8.26 5.28 4.24 GA388 28.7723.84 20.46 20.33 13.73 ND 7.26 ND GA389 28.45 23.46 21.98 17.88 12.6212.78 8.06 8.54 GA391 25.58 30.06 25.43 27.3 27.12 18.67 16.86 15.78Cyno Primary Hepatocytes-Editing % at Position 6 (Dose, Replicate #)2500 2500 1250 1250 625 625 312.5 312.5 ng/mL ng/mL ng/mL ng/mL ng/mLng/mL ng/mL ng/mL gRNA rep1 rep2 rep1 rep2 rep1 rep2 rep1 rep2 GA34640.1 37.48 36.97 34.08 25.91 25.59 14.83 15.25 GA376 29.57 29.57 27.6231.98 23.08 22.29 15.12 15.75 GA377 35.36 42.67 35.66 43.35 30.68 34.7321.24 21.79 GA380 43.27 44.85 39.03 38.76 33.31 34.1 21.83 21.51 GA38139.45 47.85 35.82 41.03 28.42 31.87 15.51 18.36 GA382 36.34 39.97 32.7437.41 26.15 25.68 14.08 12.74 GA383 39.02 53.37 35.44 44.23 28 33.42 1620.03 GA384 26.71 22.52 20.97 21.53 14.38 16.21 8.31 8.03 GA385 38.4437.36 38.31 31.84 31.97 28.48 18.23 17.68 GA386 48.11 43.61 40.09 40.4437.59 37.42 23.07 25.27 GA387 27.3 21.08 23.71 21.53 16.82 13.62 7.346.03 GA388 41.95 36.03 32.87 29.9 23.65 25.43 12.13 13.19 GA389 34.6233.47 32.15 29.86 25.87 23.29 11.17 12.33

With a view towards establishing the safety of a base-editing therapyknocking down ANGPTL3 in the human liver in vivo, a list of candidatesites in the human genome for off-target mutagenesis was assembled usingtwo different methods. The first method used bioinformatic analysis ofthe human genome, identifying all sites with a PAM sequence compatiblewith Streptococcus pyogenes Cas9 (and therefore ABE 8.8) and aprotospacer sequence with up to 4 single-nucleotide mismatches with theGA100 spacer sequence 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14).

The second method to generate candidate sites used an in vitrobiochemical assay, ONE-seq, that determined the propensity of aribonucleoprotein comprising the ABE8.8 base editor protein and gRNAGA441 to cleave oligonucleotides in a library. The reference humangenome (GRCh38) was searched for sites with up to 6 mismatches to theprotospacer sequence specified by the ANGPTL3 gRNA(5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14)), and sites with up to 4mismatches plus up to 2 DNA or RNA bulges using Cas-Designer wereidentified. More specifics on ONE-seq library preparation, experimentalprotocol, and bioinformatic analysis are described in the additionaldetailed methods section.

Oligonucleotides with higher sequence counts reflect a higher propensityfor Cas9/gRNA cleavage in vitro and represent the sites most likely tosuffer off-target mutagenesis in cells. The top candidate sites areidentified in Table 13. Validation of candidate sites was performedusing ANGPTL3 edited, and untreated human primary hepatocytes. Uponnext-generation sequencing (Table 14), when the observed base editingrates in control cells were subtracted from the observed base editingrates in LNP-treated cells across the on-target site and candidateoff-target sites (to account for background sequencing errors inherentin next-generation sequencing), appreciable base editing were observedat the on-target ANGPTL3 target site.

TABLE 13ANGPTL3 gRNA (Protospacer 5′-AAGATACCTGAATAACCCTC-3′ (SEQ ID NO: 15))candidate off-target sites identified by ONE-seq SEQPotential Off-target ID chromosome location Sequence (5′-3′) NO:alignment 1 4546642 CAGATACCATAAAAATCATCTGG 682 X60 1 6955991AAGATACTGAAAAACCCTGCAA 683 RNA41 1 16544549 CAGAGAGCAGAGTAAACCTCTGG 684X60 1 23996891 CAGAAAACTATAACCCTCAGG 685 RNA32 1 26088616AGAATGGCTGCATAACCCTTGGG 686 X60 1 27386898 AAGGTTACTTAATAAACATCTGG 687X60 1 28115884 AAGATACCTGAATTAAATCTCAAA 688 DNA41 1 30795252AAGTATCTTGAATAACCCTCCTG 689 X50 1 31542787 CAAATAACTAAATGACCCTCTTG 690X60 1 32826604 AAGATACTGAAAAACTCTCTTC 691 RNA41 1 33418548TAGATAACCTGAATAGCCCTATAG 692 DNA41 1 45361452 TAGATACTTCAATACCTCTCAGT693 X60 1 57486127 AGCAATCCTGAAAAATCCTCTGG 694 X60 1 59422147ACGATACTTGGATAACCCTCTGG 695 X30 1 59572755 GAGATACCTAATAAGCCTCTAT 696RNA41 1 62604219 AAGATACCTGAATAACCCTCTGG 697 XOO 1 66687653AAGTTACTGAATATCTCTCAGC 698 RNA41 1 76436411 AGTATACCTCAATAACCACCTGG 699X50 1 77254313 AAGATCCAGAAGAATCCCCTGG 700 RNA41 1 78181978AGGATACCTGGAAACCCTACTGG 701 X60 1 83689742 AAGATAACTAGCTAGCTCTCTGG 702X60 1 89670639 GAGATACTTGAATAAACTTCTAT 703 X60 1 93001069AAGATACCTGACTAACCTGTTTT 704 X60 1 97782174 TAGATACATGATTAAACCACTGC 705X60 1 110981606 TATATACATGAATAACTCCGG 706 RNA32 1 114341782ATTATTCTTGAATAACCCCAGGG 707 X60 1 114403243 AAGATACCTGAGAAACTCTGCAA 708X60 1 114800896 ATGACTGCTGAGAAACCCTCTGG 709 X60 1 114851709AAGAATCCTAGATATCCCTCAGT 710 X60 1 116440140 AAAATACCAAAAAAACCCTATAG 711X60 1 118751265 AAGAATTTGAATAACCCTCAAT 712 RNA41 1 119001156AGGCTAACAGAATCACCCTCGGG 713 X50 1 150002706 GTGATCCTGAGTAAACCTCAGG 714RNA41 1 156332931 TAGATACCTATAAGCCTCTGG 715 RNA22 1 162745410AAGATACATGAAAAAAACATTGG 716 X60 1 164962899 AAGATACCTTAAAACCCTTCAGC 717X50 1 165289582 AATTTACCTGAATAGTCATTAGG 718 X60 1 167577809AAGATACACAAATAGCCATCAAG 719 X60 1 170883188 AACAAACCTGAATAGTCCTACTG 720X60 1 175232700 CAGATATCTGAATCACTCTTCTG 721 X60 1 176788495AAAATATGAATAACCCTTTGG 722 RNA22 1 177848832 CAGATACTGAGTCACCCTCAGC 723RNA41 1 178104014 AAGTACCTAAATACCCTTGGGG 724 RNA41 1 179507899AAGATACCATTCTAACCCTCTGA 725 X50 1 180840385 AAGGTAACTAAGAAACCCTCAGA 726X60 1 183484429 CTCATACCACAATAACCCTCATG 727 X60 1 183743474AAGATAATGAATATCACCCAGG 728 RNA41 1 185824500 AAGATACTAATAAACACTCAGG 729RNA41 1 188687384 CAGATACCTGAATAAGCAGAAGC 730 X60 1 194817380ATAATACCTGAATACTCTCAGA 731 RNA41 1 199542263 TCTATACCTGCATAACCCTAAGG 732X50 1 201739039 AAGTTACCAGGAGGACCCTCAGG 733 X50 1 203127991AAGAGACCGGAATGCCCCCTTGG 734 X60 1 209196372 AACAGGCCATAAGAACCCTCTGG 735X60 1 209428278 TAAATGCCAGGATAAGCCTCTGG 736 X60 1 217518556TACATAACTGAATAACTCTCCAA 737 X60 1 222014072 AAGAAAGCTGAGTAAGCCCGGG 738RNA41 1 228023082 GGGATACCCGAATAACCCCACAG 739 X60 1 228546593GAGACACCTGGGTAACCCCAGG 740 RNA41 1 228724320 GTTATACCTGAATAAATCTGGGG 741X60 1 230479071 AAAATACCTGCATAACCCTGAGA 742 X40 1 230750818CAGTTCCCTGAGTCACCCTCAGT 743 X60 1 231757688 AAGATACATGAACTTCCCACAGG 744X50 1 232190288 AAAATAAGTGAATAACCCACTAA 745 X60 1 233265568TAGGTACCTGAGTAACTCTGCGG 746 X50 1 235139684 AAGAAACCAGATAACCCACAGC 747RNA41 1 238895801 AAGATACCTGCATAATCTAGGA 748 RNA41 1 244048003AGGATACCTGCGTAACTCGCAGC 749 X60 1 245228289 AAGATACCTGGAGATGCCCCTTG 750X60 2 2541015 AAGATATCAGAATAACTCTGGCT 751 X60 2 5866267CCTGTACCTGCATAACCTTCTGG 752 X60 2 11192249 AAGATACCTAAAAAGCCCCCAGC 753X50 2 14318310 TAGATACCCAAATAGCCCCTCAGG 754 DNA41 2 17555591TCTATACCTAAATAGTCCTCAGG 755 X60 2 18083124 AAAATACCAGAATAACCCATTTA 756X60 2 18570047 AAGATATCTGAATAAACTCCAAA 757 X60 2 23315873ATGATTATTAAATAACCCTCAAG 758 X60 2 24850333 AAGATCCCTGATTACCATTCCAG 759X60 2 28691746 AAGATACCTGGCTCAACCTCATC 760 X60 2 29326137AATGTGGCTGAAAAAGCCTCTGG 761 X60 2 30453369 AAGATACCTAAACATTCCAGTGG 762X60 2 31504056 AAGAGAGCTGGACAACTCTAAGG 763 X60 2 33271364AAGATAGGTCAATAACCACCCGG 764 X50 2 33829479 TAGATAACTGAATAAACTCTATGG 765DNA41 2 35540632 AAGATGTATTATTGACCCTCTGG 766 X60 2 38119450TAGAAAACAGAATGACTCTCAGG 767 X60 2 42166900 AATAACCAAAATAACCCTCCAG 768RNA41 2 46398173 TATGTACCTAATAAGCCTCAGG 769 RNA41 2 49841412CATATATCTGAATAGCCTTCTGG 770 X50 2 56159084 AAGATACAGAATTCCCCTCTGA 771RNA41 2 56257553 AAGTTACATGAAAAACATCTGG 772 RNA41 2 57130463AAAATACCTGAATAATCCTCGAA 773 X40 2 59482506 AAGATACCAGAGAAAGTCTCAGA 774X60 2 60290320 CAAATACCAGAATACCCTACAGG 775 X60 2 64169496ATGATCCTAAATAACCTTCCAG 776 RNA41 2 68986545 AACAACATGAATGACTCTCTGG 777RNA41 2 71741885 AAGATAAAGGTATCTCCCTCTGG 778 X60 2 72415642AAGCCAACTGAATAACACTGTTG 779 X60 2 72619690 ATGATACCTGAATACCTCTAATG 780X50 2 75266724 AAGCCAACTGAAGAGCCCTTGGG 781 X60 2 80321182AAGAACTTGATAAACCCTCAAG 782 RNA41 2 87443936 TTGACACCTAAATAACCCTTTGG 783X50 2 98349660 AAGAGACCAGAAGCAACCTCTGT 784 X60 2 100696913AAGAAACATGAACAGAGCCTCAGG 785 DNA41 2 104408132 GAGATACCTGTATAAGTCACTAG786 X60 2 108664444 AAAATGCCTTAAGAACCCTGGGG 787 X50 2 111506561TTGACACCTAAATAACCCTTTGG 783 X50 2 112735695 TAGATACTCAGTAACCCTCTTG 788RNA41 2 115162717 AAGAGACAGGAATAACCCTCAGC 789 X40 2 121516112AAAATACCATGTAAAACACTCTGG 790 DNA41 2 122492850 GAGATACAGAATAGCCCTGGGG791 RNA41 2 124401576 AAGAACCTAAATCACCATTGGG 792 RNA41 2 128856709AAGATATCTGGATAATCCACCAA 793 X60 2 141714040 AAGATACTGGAATAAACATAAAG 794X60 2 143719680 AAAATGCCTCAATATCTCTGTGG 795 X60 2 144191771AAAATACCAAAATAACCCTAAAG 796 X50 2 149423627 TAGATACCTGAAATACTCTATGC 797X60 2 153591411 AAAATACCATAATTTCCCTCAGA 798 X60 2 154142624AAGATACCAAATAAACCTCTGA 799 RNA31 2 155791011 AAGATACCTAAAATACCTATTGG 800X60 2 155965426 CAGATACCTGAAAAACTCTCTCT 801 X50 2 157734996AATAAACACTGAATAACCCTTTGT 802 DNA41 2 163699397 CAGCTACCTAATTACCCTCTGG803 RNA31 2 163782922 TAGGTACCTGATTTACCCTGATG 804 X60 2 166944919AAGATACCTCAGGAACCAGCAGC 805 X60 2 168821195 AAAAGACCTAAAAAGCCCTCTGG 806X50 2 169944031 TCAATTACTGAATAACCCTCTTG 807 X60 2 170941666AATATACATGAATAAATCTCACA 808 X60 2 173215359 AAAAAACGTGAAAAACCTTAGGG 809X60 2 174371138 AAGATACCTGTTAGCCCTCACC 810 RNA41 2 176698280AAGTTTCATGCATAAAACTCTGG 811 X60 2 180424100 AAGGTACCTGAAGAACTCAACTG 812X60 2 181934742 AAGATGTATGAATAACTCTCGGG 813 X40 2 183685747AAGCAACCTAAATGTCCATCAGG 814 X60 2 183737398 GAGATACCTGGATAACCACTTGG 815X50 2 184251994 AAGACACCTGGAGAACTCTCGGG 816 X40 2 188364288AAGTAACCCAATAACCCTGAGG 817 RNA41 2 188883471 ACAATACGTGAATGAATCCTCTGG818 DNA41 2 191075654 AAGATTCCAGACATACACTCTGG 819 X60 2 191371557TAAGTAGCTGTATAACCCTGGGG 820 X60 2 195665474 TAAATACTTGAAAAACCCACAGA 821X60 2 196276026 AAGTTATCTGACTAACAATCTGT 822 X60 2 196963744AATATACATGAATTTCTCTTAGG 823 X60 2 197268832 AAGATGCCTCCCTAACACTGGGG 824X60 2 197475333 TACATACCAACAACCCTCTGG 825 RNA32 2 198342120AATAGAGCTGAAAAACCCTCACT 826 X60 2 198435701 AAATTAGATGAATAACCCTTAGT 827X60 2 202129138 AAAATACCTGATACCCCCTGG 828 RNA32 2 202797188AGAGTACCTGAATACCTCTGG 829 RNA32 2 203165696 AGAATATCTGAATTACCCTCAAG 830X50 2 204928058 AAGATACATGAGTAAACCCAGC 831 RNA41 2 206764957AGGAGACCTGCACAGGCCTCTGG 832 X60 2 216172775 AATTTATATGAAAAACCCACTGG 833X60 2 217541307 AAGATACCTGATCAATCCTCTTC 834 X50 2 217737466AAGATACCAGACCAAAGCTCAGG 835 X50 2 220751309 TAGAGACATGAAAAACCCTCCAA 836X60 2 221762944 AATATTCCTGAATCCCTCGGG 837 RNA22 2 226128392CTCATACCTGAATAACCTTCTCA 838 X60 2 226932531 CAGATACCGAGAAAAACCCTCTGG 839DNA31 2 227560446 AAGATATATGAAAAAGCCTATGC 840 X60 2 228586979CAGATACCTCACCAACACTCCAG 841 X60 2 232730167 CAGAGACCTGAAAAGCCCTGCCG 842X60 2 240208622 AAAAGGCCTGCAGAAACCTCTGG 843 X60 2 241728292CAAATCCTTGAATCACCCCCAGG 844 X60 2 241778506 AAGATACCTGGCCACACCCCAGG 845X60 3 539550 GAGATAATGAATAGCCCTCCGG 846 RNA31 3 7611430AAGATACCTGTCATCCCTCCGT 847 RNA41 3 8738135 GGGATACCTGCATAACTCTCAGG 848X40 3 11156153 GAAATGCCTGAATCACACTCGAG 849 X60 3 11683514AAGATACTGAATCAACTTCAGT 850 RNA41 3 16474484 AAGATACCAGTACACCCCCAGG 851RNA41 3 19871463 ATGATACCTCAATAACCCTATTT 852 X50 3 25070750AAAATACCTGAACATACCTTGAG 853 X60 3 35461847 TACATACCTAACAACCCCTCAGG 854X60 3 35699317 AAGAAACAAGGGAAACCCTCAGG 855 X60 3 36482333AAGATACCAGGATGGCTCTCTGC 856 X60 3 37570248 CAACAACCAGAATAACCCACAGG 857X60 3 37680767 AAGATTAAAAGAATAAACCTCAGG 858 DNA41 3 38875399AACTTACCTGCAAAACCATCTGG 859 X50 3 56933123 AAGATCTGAATTACCATCAGT 860RNA32 3 61401423 GAGATCCTTAACAACCCTCAAG 861 RNA41 3 64532843AAGATATCTGAGCCACACTCTCG 862 X60 3 68532737 AAGCTACCTGAAAACCTTCAGG 863RNA21 3 70442317 AAGATGACTGAAGAAACCTTGGG 864 X50 3 70620477AAGATCCCTGCAAACCCTTTTGG 865 X60 3 71429771 AAGACAGCTAAATCACCCCACGG 866X60 3 71714408 AAGATCACAGAATCACCCTCCTC 867 X60 3 74054464AATAGACATGAATAACCCTAAGA 868 X50 3 76238395 CAGACACCTTACTAACTCTCCTG 869X60 3 82942470 AAGATACACAAATAAACTTCTAG 870 X60 3 83311768AAGATATCTGAAAAACTCTCAAA 871 X50 3 84371314 AAGACACTGAATAACCCACAGA 872RNA31 3 86668690 TAGATAGCAAATAACCCTAGGG 873 RNA41 3 98032158AATAGACCTAAATAACCTCAAG 874 RNA41 3 99319950 AAGTTACCTGACTAACCCTGAGG 875X30 3 100809940 AAGATATCTGAATAACTACAAGG 876 X50 3 107069791AAGATACTTGGATAACTCTGCTA 877 X60 3 107509328 AAGAGGCTGGAATAAGCCACGGG 878X60 3 111016912 AAGATACATAAATATCACTGCAG 879 X60 3 118950239CACTAACCTGAATAGCCCTAAGG 880 X60 3 120988045 AATATACCTGAAAAACCATCAGG 881X30 3 121667776 AAAATACCTAAAGAAATTTCAGG 882 X60 3 122037269CGTGTACCTGAAAAAGCCTCAGG 883 X60 3 126759212 GTGAGGCCTGAAGAACACTCTGG 884X60 3 133985020 AAGTTACTGAATATCCCTCCAG 885 RNA31 3 140863741AAGAAACCGGAATGAGCCCCCAG 886 X60 3 141050688 CAGATACCAGAAGCACCCTCGAG 887X50 3 142685343 TAGATAACTGAAATATCCCTCTGG 888 DNA31 3 143875802GAGATACCCAAATAAACCTCAAA 889 X60 3 151175376 AAGATACTGAATAAAGCTCTTA 890RNA41 3 151226712 AAGATAACTGAAGAAACTGAAGG 891 X60 3 151395707AAGATACCAGGGTAACTCTATGA 892 X60 3 155083487 AAGATACCTGAATATCCAACCTA 893X50 3 161319971 AAGCTGACTGAAGAGCCCTTGGG 894 X60 3 168443226AGTATTTCTGACAAACCCTCAGG 895 X60 3 169534358 GAGATAGCTAATAACCCCCTGG 896RNA31 3 171933625 AAGACAACTGAAAAACGCTGTGG 897 X50 3 173995167AAGACACCTGAATAATCTCTGGA 898 X60 3 176890998 ATGATAAGTGAATAAGTCTCATG 899X60 3 177768689 AGGATATATGAAAAAGCCTGCGG 900 X60 3 187379619AAGATACCCTAAAAGCCTTTGG 901 RNA41 3 188484179 AAGATGCATGAATAACCTCTGGA 902X60 3 190699564 ATGACACCAGAAAGACCCTAAGG 903 X60 3 192710228AAAATACTAAATAACCCCATGG 904 RNA41 4 723653 CAGATTCCTGGAAGACCCTCAAG 905X60 4 11334933 AAGAGACCTCAAGGTCTCTCTGG 906 X60 4 12971711AAGACTAAAGAATAACCCTCTGG 907 X50 4 15327544 AATATGCCTACATTACACTCAGG 908X60 4 16411507 AAGACACTGAATAACCCTTGGC 909 RNA31 4 20157051AAGATAGTGAGTAAGTCTCAGG 910 RNA41 4 23244775 TAGATAAGAATAGCCCTCAGG 911RNA32 4 26200297 TCTATACCTCAATAACCTCCTGG 912 X60 4 28742386AAGATACCTAAGTGCTCCACAGG 913 X60 4 29008961 AAGAAACCTACAATAACCTTCATG 914DNA41 4 33579022 CCGATATCAGAATATCCCTTAGG 915 X60 4 44748785AAGATGCATGAAAAAGCTTCAGT 916 X60 4 44812393 TAGATATCTGCAGAACTCTCCGC 917X60 4 45258647 AACATACATCAATAACTTTCTTG 918 X60 4 46495164AAGATACTTGAATACCAATCAAG 919 X50 4 47192935 TAAAGACCTGAATAACCTCAAG 920RNA41 4 47840228 AAGATATTGAATATACCTCAGG 921 RNA31 4 51965380TAGTTACCTGAATACACCTCTGG 922 X40 4 66291743 TATATACCTGGAAAACCCTAAAG 923X60 4 74170265 TAGATACATAATAACCAACTGG 924 RNA41 4 84374124GAGATACTTGAATCCCTCAGG 925 RNA22 4 87976264 AAGATACCTGTATGACACTGTGT 926X50 4 88117912 AAGATACCTAAATAACAAGCTGG 927 X40 4 90779595AGGATACATGCATAACCCATGAG 928 X60 4 91244542 AAGATCCAGAATAATTCTCTGG 929RNA31 4 91936467 AAGATACATGAAAAATTCTCTCA 930 X60 4 93373949AATATACCCTAATAAGCCTTCTG 931 X60 4 93711948 AAGATGGCTGAATAGAACCTTCCAG 932DNA42 4 96659665 AATATACCTTATTATCCTTCAGA 933 X60 4 100450684CAGATACAAGAGTACTCCTCAGG 934 X60 4 101268626 TCTATACCTAAATAAACCTAAGG 935X60 4 104738004 TAGATACCTCAATAATTCTCTCT 936 X60 4 115433239AAATAACATAAATAACCCTCAGA 937 X60 4 115953135 CAGAAGCTGGATAGCCCTCGGG 938RNA41 4 136101368 AAGAACATGAGTAACCCTAAGG 939 RNA31 4 136644335AAGATACTTGAAAAACACCAGGT 940 X60 4 136853658 AAGAGAACTGAAAAGCCTCAAG 941RNA41 4 144234750 AAGATACCAAATAACTCTCTGG 942 RNA21 4 146835606AATATGCCTGTAACCCTCAAG 943 RNA32 4 149064984 AAGCTGACTGAAGAGCCCTTAGG 944X60 4 155932126 AAAATTCATGAAGAACCCTGGGT 945 X60 4 157312624AAGATACGTGAATAACTCAGGAT 946 X60 4 159332385 AAGATATCTGAACAATTCTAAGG 947X50 4 160192361 AAGATACTTGAATCACTGTCAGT 948 X50 4 168412887AAGATCCTGAATAATTCTCTGG 949 RNA21 4 170886954 AAGAAGCCTGGATAGAACTCTGG 950X60 4 172543878 TTGATACCTGAATAACACTCCAG 951 X40 4 172763007AAAAAAAATGAATTACCCTCATG 952 X60 4 174561024 GAGATCCAGAATTACCCTTGGG 953RNA41 4 181671436 AACATACCTAAATAACTCCTGGC 954 X60 4 184625030AATCTACCTGATGACCCTCGGC 955 RNA41 4 186075007 AAGTTAACTGTAAAACAGCCTCAGG956 DNA42 5 6252615 AAGAAATCTGAAAGCCCTGGGG 957 RNA41 5 7102627GAGATACCTGCATAACACTTAAT 958 X60 5 7274614 CAGATAACTGAACAACCAGATGG 959X60 5 7657678 AACTTAGGTGATTAACCCTCACG 960 X60 5 8704833ATGATACTGAATAAGTCTCAGG 961 RNA31 5 9774717 AAGATCCAGAATAATTCTCTGG 929RNA31 5 13575913 AGAATACCTAGTGACCCTCAGG 962 RNA41 5 16123391AACAGACCTTCATAGCCCTCAGA 963 X60 5 16404802 TATATACCTAAATAACCTAGGG 964RNA41 5 20733472 AAGATCTCTGAAATGCCCTGAGG 965 X60 5 23248793AACTTAACATAATAACCTTCAGG 966 X60 5 34351741 ATGATAGTGAATAATCCTCATG 967RNA41 5 35773444 AAGATACGAGAATAACCTCAAGA 968 X60 5 35946321AAACTACCTGGATGACCCTCTGA 969 X50 5 36662538 AAGAAGATGAATTACTCTCTGG 970RNA41 5 42904833 AAGATACAGAGTAACTCTTGGG 971 RNA41 5 43693099GAGATAGCTGAATAATCCCTCAG 972 X60 5 45541710 AAGATACATCAGTAAGCCACAGT 973X60 5 55453226 AAAATACATTAATAAAACTAAGG 974 X60 5 58609559AGTAGAGGTGAATAACCCTCAGC 975 X60 5 60295257 CAGATACTTAAATAACCATAAGG 976X50 5 61555769 AAGAACCTGACAAGCCCTCAGA 977 RNA41 5 66999062AAGATCCATCAATAAACCACGTG 978 X60 5 69412657 AAGATACTTTTAAAACCCAGTGG 979X60 5 73013090 AAGATCCTGAATTACTCTCAAG 980 RNA31 5 73504738AAGATACCTGAACAACTCCACAC 981 X60 5 75225969 AAGCTGACTGAAGAGCCCTAGGG 982X60 5 78444765 TTGATACCTGTATTACACTCATG 983 X60 5 82799696AAGATACAGAGAATAACCCTCACA 984 DNA41 5 83300276 AAAATACCTCACTAGCTCTCCAG985 X60 5 85150663 AAAATAATTGAAAAACTCTCTGT 986 X60 5 87098398ATAAGAACAGAATAACCTTCTGG 987 X60 5 91842929 AAGAGTCATGCATAGCCCTCCAG 988X60 5 105774459 AAGATACTGAATAAACTCCTGC 989 RNA41 5 106733933AAGAAACACAAATAACCATCAGA 990 X60 5 108874504 GAGATGCTGAATAACTCTCTGG 991RNA31 5 112311434 AAGATACTGAGTATCTCACAGG 992 RNA41 5 112422645AAGACACCAAGATAGCACCCTCGGG 993 DNA42 5 113833519 AGTATACCTTAATACCCCCTGG994 RNA41 5 114129926 ATTATACCTAATAACCCTATGG 995 RNA31 5 117372593AATATAGCCCAATATCCCTCATG 996 X60 5 118452920 CAGATACCTGAATGATCCTTTTC 997X60 5 119760952 AAGAAAGCTGAGTAGTCCTCAGA 998 X60 5 132792147AAGGGACAGGAAGAACCCTGAGG 999 X60 5 132904762 AAGATAGCTGTATAACCTTTATC 1000X60 5 137163670 AAGTTAGCGGAACTCACCCTCAGG 1001 DNA41 5 137764269AAGAGAGCTAGCTAGCCCTCTGG 1002 X60 5 142640990 AGGATTTCTGGATAACACTTTGG1003 X60 5 143734240 GAAGTACCTGAAAAACCCTCTGC 1004 X50 5 148112086GAGGTACTTGAATCACCATCAAG 1005 X60 5 151722432 TAGCTTACTGAATAACCCTAAGG1006 X50 5 152708508 AAGAGGCATGAATAACCCTGTTC 1007 X60 5 153529703CAGTTACGAATAACCCTCAGA 1008 RNA32 5 156251323 AAGATACTGAAAAACCCTCTGA 1009RNA21 5 160330029 AACAGACATGAAGAAGGCTCAGG 1010 X60 5 163829739ACCATACCTGAAGGACCATCTTG 1011 X60 5 164401431 AAGAGACCTTAAAACTCTCTGT 1012RNA41 5 165782539 ACAAGACCAAAATAACCCACAGG 1013 X60 5 172047699ACGATGGCAGAACAAACCTCAGG 1014 X60 5 173042104 AAGATACCTCAAACACCAGCCGT1015 X60 5 175751428 AAGACACCTAAATATCCCTGGGA 1016 X50 5 177550455AAGATACTGACTAATCATCTGC 1017 RNA41 6 2810238 TAGACACCTGAGTCACCCTCTTA 1018X60 6 3633347 GTGATACCAGATTAACCTCTGG 1019 RNA41 6 3993855AGAGTAACTGAATAACCCTACAG 1020 X60 6 6543792 TTTGTACCAGAATAACCCTCCTG 1021X60 6 8845584 AAATTAACTGCATAACCCTGGGT 1022 X60 6 12146644AAAAAACTTGAATTACCTTCTAG 1023 X60 6 13274573 CTGAAACCTAAATAATCCCCCGG 1024X60 6 15494007 AAGACCCAGGAATCACACTCTGG 1025 X60 6 16159742AGAAGACCTGAATACCCCCAGG 1026 RNA41 6 17837365 CAAATACCTAAAGAACTCTCCTG1027 X60 6 18241159 CCTATACCTAATAACCTTCAGG 1028 RNA41 6 20501819AAGATACCTGGAAAACTCCCAAA 1029 X60 6 22811364 TAGACACCTAATAATCCCCAGG 1030RNA41 6 24130572 GTGATACCTGTCAGACCCTCGGG 1031 X60 6 25968782AAGATACCAGAATTCCCCTCTTC 1032 X50 6 26726581 AAGATGGCTGAGAAAAACTCAGG 1033X60 6 26775081 AAGATGGCTGAGAAAAACTCAGG 1033 X60 6 28642665CGTGTACCAGAATCACCCTCAGG 1034 X60 6 28976992 AAGAACTTGTAGAACCATCAGG 1035RNA41 6 31728324 AAGATACCCCATCACCCCTCCCG 1036 X60 6 31925117AAGATCAGCAGAAGAACCTTCTGG 1037 DNA41 6 32569518 AGTATACCTAAACAAACCTCAGA1038 X60 6 35414348 AAGATCCTGCATACCCTTCTGT 1039 RNA41 6 39979448AAGATCCAGATTAACCCTCTAG 1040 RNA31 6 40805097 AACATCCCTGAAGACCCTTGAGG1041 X60 6 41733389 AAGATACCTGGGTCCCCATTTGG 1042 X60 6 42264231AAGATCCTTAAATACCCTCTGG 1043 RNA31 6 42676866 AAGATAACTAACTCACCTTCCCG1044 X60 6 45506487 ATGATATATGAACAACCCTCTGG 1045 X40 6 45565147AACATACTCGAATACCCCTCAAA 1046 X60 6 45812353 AGCTTACCTGCTTAACCCTCAGG 1047X50 6 45989719 AAGATACCAAAATTCCCCAATGG 1048 X60 6 46206185ACGATACTGGAATAAGCCTTTGA 1049 X60 6 51499203 CAGTACCTCAACAACCCCCTGG 1050RNA41 6 51857715 AATATCCTGAATAAACCTCTAG 1051 RNA31 6 57915493AAGACACCTGATAAAAACTCTGT 1052 X60 6 62452292 AAGATACAGAATAAACCTAAGG 1053RNA31 6 66308858 GAGATACTTGAACAACTCACTGG 1054 X50 6 66899926AAGAAACCAGAGAAACCTTCGAG 1055 X60 6 71004311 AAAGTACCTAAATAACCAACCAG 1056X60 6 76467643 AAGAAGGTGAATAACACTTAGG 1057 RNA41 6 78402401AAGATACCTGGCTAGCCATATGC 1058 X60 6 79368236 AAGCTACCAGAGGAACAATCAGG 1059X60 6 84651006 CATTTACTGAATAACCCTTTGG 1060 RNA41 6 84877021GATAAACATGAAGGACCCTCAGG 1061 X60 6 89636030 AACGTACCTGAACAACCCTCAGG 1062X30 6 90026983 ACACTACCTGAATGACCCTCAAG 1063 X50 6 91316610AATATACATGAAACACACTTAGG 1064 X60 6 100261928 AAGATACCTAAATGTCCATCAAC1065 X60 6 101477606 CAGATATGAATAAACCTCTAG 1066 RNA32 6 104427293AATAGTACTCAATAAACCTCTGG 1067 X60 6 105452361 AGTATACATGAATAGCCCTCAGG1068 X40 6 113288211 TTGCTACCTGAATAACCCTCTTG 1069 X40 6 125169983AAGATATCTGAGGAAGCGTCTTG 1070 X60 6 125494646 AAGATACCTGAAACCCTCTGG 1071RNA02 6 126401280 CAGATATTGAAAAACCCTCTGG 1072 RNA31 6 127951944AAGGTATCCAAATAACCCTTTGG 1073 X50 6 133766716 TAAATACCTGAACAACTCTAGGG1074 X50 6 134020048 AAGATTACATGAGTACCCCTCCTG 1075 DNA41 6 141080346ATGATAGTGAATAACTCTCTTG 1076 RNA41 6 141657228 AAGATATCTGAAAAATCCACAAA1077 X60 6 142866995 AAGATTTCTTATAACCCTCTGG 1078 RNA31 6 146355335AAGATACTCTGAATAACTTTCTGG 1079 DNA21 6 147553748 AAGTCATCTGAATAACCCTCAAG1080 X40 6 152311384 AAGATACGTGCAATAACTATCAAG 1081 DNA41 6 158194025AAGATACATTAGTAACATTTTGG 1082 X60 6 159249441 AAGAAACACTTAAAAACCATCTGG1083 DNA41 6 159877092 TAGATACCTGTATAACCACCACC 1084 X60 6 163714704AACTTACCTGAACATGCCCCTGG 1085 X60 6 166082374 TAGATACATGGATTAACTTCTGG1086 X60 6 168015970 AAGACAGCAGTAAAACACTCTGG 1087 X60 6 168145686AAGATCTGGAAGAACTCTCTGG 1088 RNA41 7 5143278 AAGAAAGGTGAGCAACACTCGGG 1089X60 7 5180429 AAGATACTTTAAAAAACCCCTGC 1090 X60 7 12098480AATATACCTAGCTAAGCCTCAGA 1091 X60 7 16189181 AAGATAGCCTGAGTCACACTCTTG1092 DNA41 7 17175165 AAGAAAACAGCGTAAGCCTCAGG 1093 X60 7 18301478ATGAAACCTAAATAAGTCTCTGG 1094 X50 7 18326887 TGTTTACCTGAATAACCTTCAGG 1095X50 7 20063992 AAGATATCTGAAAAGCCCACTTT 1096 X60 7 21393775TGTAAACCTGGATAACCCTCTAG 1097 X60 7 22673635 AAGTATCATGAATACCCTCAGG 1098RNA41 7 25237966 AAGATCCTATATAACCCTCTGA 1099 RNA31 7 28451369AAAATACCTGAATAACCTGGGCC 1100 X60 7 36309411 AAGTTACCAGAACAACCCTTAGG 1101X40 7 43099959 AAGAATTTGAATAACTCTCTAG 1102 RNA41 7 45063943AGGATACCTGAATCCCTCCAG 1103 RNA22 7 46108303 GAGACACCTGAATAAGTGTCTGG 1104X50 7 46457470 AAGCTAGTGAAAAACCATCAGG 1105 RNA41 7 47819173AGGATACTTGAATATCCCTGCCT 1106 X60 7 49066063 CAGATACTGAAAAACACTCTGG 1107RNA31 7 49306954 AAGAAATCTGGATAACCCCCAAT 1108 X60 7 60892285AACATACCCAAAAAAACCTCTGA 1109 X60 7 61597579 AACATACCCAAAAAAACCTCTGA 1109X60 7 62026418 AACATACCCAAAAAAACCTCTGA 1109 X60 7 78658625AAAATATCCAAATAACCCCCTGG 1110 X50 7 81886792 AAGATATCTGAATAATCATCAGG 1111X30 7 88664778 AAGATACTGAGTAACACTGAAG 1112 RNA41 7 89093321AAGTTACCTAAATAACTTTGG 1113 RNA32 7 89117479 ACCATAATTGATTAACTCTCTGG 1114X60 7 94742185 AAGAAACCTAAGTAAACCTCTAA 1115 X60 7 96159184AAGATACATCATCACCCTCCTG 1116 RNA41 7 96499495 AAGACACATAAGGAACCCTCATG1117 X60 7 101395824 GAGATACCTGAGCTGCCCTCAGC 1118 X60 7 104765414GAGATACCTAAATAACCTTCCAG 1119 X40 7 107527319 AAGATACGAATAATCCTCCGA 1120RNA22 7 114126788 ATTAAACCAAAATAACCCTCGGG 1121 X50 7 117090821CACAAACCTTAATAACCCTCAAG 1122 X50 7 117393513 AAGATAAAAAAATACCCCTAGGG1123 X60 7 123985680 AAGATATCTGAAAAAGTCTTGAG 1124 X60 7 124105262GAGATAACTGAATAACCATTTGG 1125 X40 7 125205185 AAGATATCTGTAAAACCCTTCAG1126 X50 7 125899502 AACATACATGAAAAATCCTCAAC 1127 X60 7 127268120AAGGTAACTGGATAGCCATCTGC 1128 X60 7 132695120 AAGAGACCTGAGTCACCCTGTCT1129 X60 7 134060777 AAGATAAATGAATTTCCTTCTGT 1130 X60 7 140628498AAGATACGGGAAATCCCCTTTGG 1131 X60 7 146763269 CAGATATCAGATAACCCTATGG 1132RNA41 7 153436410 CAGATGCTTGTAAAACCATCAGG 1133 X60 7 153479243ACAGTAGTTGCATAACCCTCTGG 1134 X60 7 155749430 AAGATTCAGCACTAACCCACAGG1135 X60 7 157980250 GAGATACTGAATTACCCTCTGG 1136 RNA21 8 4038549AAGAGACATGCATAATCTTCAGG 1137 X50 8 5643554 AAGTAAACTGAATACCTTCTGG 1138RNA41 8 7420667 AAGAAACCACTATAACCATCAGT 1139 X60 8 7890852AAGAAACCACTATAACCATCAGT 1139 X60 8 19687879 CAGATACTGTATAACCCTCAGA 1140RNA31 8 21016005 AATGTAAATGTATAACCCTCTGT 1141 X60 8 29936242AAGTTAACTGTAAAACAGCCTCAGG 956 DNA42 8 36495615 AGGATACCATAATAACCAGCATG1142 X60 8 43072291 GCAATACCTGAATAACCCATTGT 1143 X60 8 47579895AAGATACCAGACCATCTCCCAGG 1144 X60 8 49597827 AAGATCTGAATAGCACCCTGG 1145RNA32 8 55071567 AAGATCACAGGACAACCCTCCGC 1146 X60 8 55401326AAGATTCATGATAACCATCATG 1147 RNA41 8 60813307 CAGGTACCTGGAGAACCCTAGAG1148 X60 8 61779630 AAGATACCTGAACACCCTACTGG 1149 X40 8 64267393TAGCTAGCTGATAACACTCTGG 1150 RNA41 8 65828227 ATTATACTGAATAACCTTCCAG 1151RNA41 8 66202960 AAGTTACCTGCATCAAACTCATG 1152 X60 8 66947451AAGATACAGAATAACCCTCTAG 1153 RNA21 8 67331610 GATATACTCTAATAACCCTCAAG1154 X60 8 67691390 AAGATACATAAATCACTTTCCAG 1155 X60 8 67694023AAAATGCCTAATAGACCTCTGG 1156 RNA41 8 68072298 AAAACACCTGAAAAATCCTCCAC1157 X60 8 73416526 AAGAAACACTGAATGTCCCTCTTG 1158 DNA41 8 74109927GTGAAACCTAAATAACCCTCAGA 1159 X50 8 75900398 AATATACCAGAATAGTTCTCAGT 1160X60 8 76479079 AAGATACTGAATATTCCTCAGG 1161 RNA21 8 78222169GAGATACTTGAATAACCATCTCA 1162 X50 8 80098340 AAAGTACATTAAAAACCTTCTGG 1163X60 8 80176030 AAGATAATTGAATAGCCATCTTA 1164 X60 8 80682169AACAAACCCGTAAAACCCTCACG 1165 X60 8 83187784 AAGATAGTTAAATATCCCCCTGT 1166X60 8 88225407 AAGATAATAAATAACCCTCAAG 1167 RNA31 8 91354172AAGCTACTTGATAACCCTATGG 1168 RNA31 8 102629148 ATTATACCTGAATCCCTCAGG 1169RNA22 8 106844104 AAGTTATCCTAATAACCCTATGG 1170 X50 8 106983770ATGTTTACTGAATACCCCTCTGG 1171 X50 8 108954887 AAGGGACCTGAAATTCCCTGAGG1172 X60 8 115458181 TAGATAACAGAATGACTCACAGG 1173 X60 8 119894846AAGATTCTGAATAGACCCCAGG 1174 RNA41 8 120688940 AACATACCCAAATACTCTCAGG1175 RNA41 8 121544316 AAGATACCTCAATAAGACTCGGG 1176 X30 8 124209633TAGATACTGAAAATCTCTCAGG 1177 RNA41 8 124951793 AGAATACAAGAATAACCCTGGGG1178 X50 8 125028680 AAGATACCTGAAATTCCTGCAGA 1179 X60 8 125768967CTGGTACTTAATAACCCTCAGG 1180 RNA41 8 130261595 GAGATACCTAACACACACTCTGG1181 X60 8 139484036 ACAATACCTGAAAAACTCCCTTG 1182 X60 8 140142948CAGATACATTAAAACCCTCTGG 1183 RNA31 8 141381027 CAGAACCCACAATAAGCCTCTGG1184 X60 9 1548217 AATAACATGAATAACACTCAAG 1185 RNA41 9 2362898AAGTAACCCAAATAACCCCAGG 1186 RNA41 9 3085581 AAGATACAGAATTTCCCTTTGG 1187RNA41 9 7499375 AAGGTCCCTGAGCCACCCTCCAG 1188 X60 9 7701793AAGATCTGGAACAACTCTCTGG 1189 RNA41 9 9668247 AACACTATTGAATAACCATCTGG 1190X60 9 13025629 GGGATACCTGAATATTCTCTGG 1191 RNA41 9 13825298AAGATGCCTGCACATCCCTCCTT 1192 X60 9 15566128 AAGATACATGAATTACCAAAAAG 1193X60 9 18157881 AAGATGCTTGAAGTAGCTTCAGG 1194 X60 9 21582692TAGATACCTCTTATAACCCTCCAG 1195 DNA41 9 22799849 AAGATACATCAATAAACCTGATT1196 X60 9 22914887 AAGATAGTCCTATAACTCTCTGG 1197 X60 9 27875283AAAATTAGTGGATAAACCTCTGG 1198 X60 9 28235239 AAGATCCTGAAGAATTCTCTGA 1199RNA41 9 29142022 AGAATACCTGAACAACACTCTAG 1200 X50 9 33293321ATTAAACCTGAAACCCTCAGG 1201 RNA32 9 33907178 AAGATAGCTAAATAACCTCTGA 1202RNA31 9 36483605 GAGACAACTGATTATCCCTCAAG 1203 X60 9 39480043AATTTACCAGAATAGCCCTCTGA 1204 X50 9 66885197 AATTTACCAGAATAGCCCTCTGA 1204X50 9 74963383 AAGATACTGAAATACCCCTATGG 1205 X50 9 81338482AAGGTAAATGAGTAATCCTTTGG 1206 X60 9 82995745 AAGATTACTTGAAGAAGCCTCTGA1207 DNA41 9 85121255 AAAGTAGCTGAATAAACCTCAAG 1208 X50 9 88846233AAGATAACTGAAATGCCTTCGAG 1209 X60 9 89929285 ACTATTCCTGAATAACCCTTTGG 1210X40 9 94468528 AACATCCTGAATCAACCTCTGC 1211 RNA41 9 96771534GAAATACTGAGTACCCCTCAGG 1212 RNA41 9 97299066 TAGATCTAGAATAAGCCTCTGG 1213RNA41 9 100767516 AAGATACCCAAATAACCATCAGA 1214 X40 9 107058386AAGACAGCTAACTAACTCTCGGG 1215 X50 9 107981569 AAAGTAGCTGAACAGCCCTCAGG1216 X50 9 109732394 AAGATACCTCATTAATCATCACA 1217 X60 9 110545175AAGATATGGAATCAACCTCAGG 1218 RNA41 9 112078020 AAGATACCTAATAACCACAGGG1219 RNA31 9 114633862 ACAGCACCTGAATCACACTCTGG 1220 X60 9 114864429AAGATACAAAAACAACCGACTGG 1221 X60 9 114970717 AAGATACTCAATAAACCTCAAG 1222RNA31 9 116496855 ATGATAGTGAATAATTCTCAGG 1223 RNA41 9 123598985AAGACACATAAACCACTCTCAGG 1224 X60 9 125297490 AAGATACTGGATAACCTTCCAG 1225RNA31 9 125378327 ATGATAATGAATAACCCTTGGG 1226 RNA31 9 125911093AAGACACTGAAAAACCCTCAAG 1227 RNA31 9 127691320 AAGATAGCAGATTAGTCCTCAGT1228 X60 9 127974450 AAGAGATCGGGATAAGCCTTTGG 1229 X60 10 7147046TAAGTACCCAAATAAGCCTCTGG 1230 X60 10 7993040 AAGATATCTGAATATGGACCAGG 1231X60 10 11705444 AAGATAGCTAAGAATCCCTCCAG 1232 X60 10 13360457TAGTTACAAGAATAACCCCAGG 1233 RNA41 10 14735911 TAGATACATAATAGCCCTTTGG1234 RNA41 10 15941531 AAGAGCCCTGAATAAGCTTTAGG 1235 X50 10 16541599AAGATACTGAATAAACCCGAAG 1236 RNA41 10 21114219 AAGATATCTGGATAATCCCCAAA1237 X60 10 27972145 AATATACCTTGAATACCCCTCCCT 1238 DNA41 10 32570562GGGATACCTTAACAACCCTGTGG 1239 X50 10 35982871 AAAATACCTGACAGAACCACTGG1240 X60 10 38157649 AAAATACCTGAGTGACCCTAGAA 1241 X60 10 47125548AAGATTTGTGAATGACCCTAATG 1242 X60 10 50305665 AAGATACCAGTATACTCTCTGG 1243RNA31 10 51383143 AAGATCCCTGAACAACACCAAAG 1244 X60 10 54571271AAGATACCAGTCCCACCCTCTTG 1245 X60 10 55277105 TAGATACTCTCAATGACCCTCTGA1246 DNA41 10 59699528 AAGTTACTTAAATAGCTCTCTGC 1247 X60 10 62667353AAGATGCTTGAATTTCCCTAATG 1248 X60 10 82421522 AAGAGATCTGGATCACCCCCAGC1249 X60 10 83225274 AAGATATTTGAATATTCCCTAGG 1250 X60 10 84218985TAATTACCAGAATAACCCTATGA 1251 X60 10 90306612 AAGATACCTGAAATACCTGGGT 1252RNA41 10 94280573 AAGGTACCTGAGTAAACATTCTG 1253 X60 10 102517636AAGATACATGAATGAATTCAGG 1254 RNA41 10 102734271 CAGATACCTAAGAACCCTGACG1255 RNA41 10 106248382 AGTATAACAGAATAACCCTATGT 1256 X60 10 118171466AACATATCTGAATATCTCTCTAA 1257 X60 10 119789592 AAGATCCTGCCTCAACCTCTGG1258 RNA41 10 120632845 AAGATACCAAGCTAATCCTCTGC 1259 X60 10 121862898GAGATTTCTGAATAACCCACCCT 1260 X60 10 121868681 GAGAGGCCTGAGTAGCCCACGGG1261 X60 10 122274421 AAGGAAACTGGAGAACTCTCCGG 1262 X60 10 130321175AAGATACTTGAATAACCATTTCA 1263 X50 10 131549720 TGCTTACATGAATAACCCTCCAG1264 X60 11 2376222 TCTGTACCTGGATAACCCCCTGG 1265 X60 11 3277015AAGGTAACTGACAAACCATCTGA 1266 X60 11 3322831 AAGGTAACTGACAAACCATCTGA 1266X60 11 3502236 AAAATAAATGAATAAAACTGTGG 1267 X60 11 4864723AAGTCACCAGTTTAACACTCAGG 1268 X60 11 7440757 AAGATACCAGAATAAATCATTCTAG1269 DNA42 11 12052022 TAAATACATGAATAACCATTAGG 1270 X50 11 12138824AAAAGCCCTGATCAAACCTCAGG 1271 X60 11 12410906 CACTTACCTGTATAACCTCAGG 1272RNA41 11 14162519 AAGATACTTGCAGAACCATCTGA 1273 X50 11 15458757AAGATCTGAATTTCCATCAGG 1274 RNA32 11 22689538 AAGATATACATACTAACCCTCTGG1275 DNA41 11 22781693 GAGTACCATAATAAACCTCAGG 1276 RNA41 11 22831675AAAGAACCTAAATAAGCCTCTTG 1277 X60 11 25615424 AAGACATTAAAATAACCCACAGG1278 X60 11 25752459 AATATACCTGGAAAACCCACACC 1279 X60 11 26574272AAGACACATGAATATCACTAATG 1280 X60 11 26949218 AAGATATTGAATAACCCACATT 1281RNA41 11 26997914 AACATCCCAGAATGACCCACAGC 1282 X60 11 28826023AAGAAAGCTGGCTAGCTCTCTGG 1283 X60 11 32956605 AAGATAACTGACTTGACCCTGTGG1284 DNA41 11 36609914 AAGATATGAATAAAACTCTGG 1285 RNA22 11 50574461AAGCTATCTGAGAAACCCTTTTG 1286 X60 11 58705310 CAGATACCTAATAACCACACGG 1287RNA41 11 63223967 AAGATACCTGAAATTATTTCAGG 1288 X60 11 79064018TTGATACCTAAATACCCTGTGG 1289 RNA41 11 80186841 TAAATACATGAATAATCCTCGGG1290 X40 11 81449300 AATTAACCTACATAACCCTCAGT 1291 X60 11 83977145AAGATTCTTAAATAACCCACTTT 1292 X60 11 87592156 TAGAGACATGAAAAACCCTCCAA 836X60 11 88339518 AAGATTTAAGAATGAACCTCAGG 1293 X60 11 88892935AAGATACAGAATTTCCCTTTGG 1187 RNA41 11 92568878 TGGATACCTGTCAACCCTCTGG1294 RNA41 11 101689415 AAGATCCGTGAATAAACCACAGC 1295 X50 11 104364397AACATACACGAATACCTCACAGG 1296 X60 11 106039497 AAGATGATAAACAACCCTCTGG1297 RNA41 11 109887670 TAGATCCTAATTAACCCTCAGA 1298 RNA41 11 114155888AAGATACAGGTGTAGCCCTCCTG 1299 X60 11 117119305 CTGATATGTGAAAAACCCTAAGG1300 X60 12 3649402 AACATACTGAACCAGCCTCTGG 1301 RNA41 12 3696467AAGACACGAGAAGAACCTTCTGA 1302 X60 12 3830100 AAGACACTTGAATACCACTCTTT 1303X60 12 4704350 TACATACCTTGATAACCTTCAGG 1304 X50 12 8120901AAGTTACCTGAACAAACCTGCAA 1305 X60 12 9334202 AAAAAACTAGAAGAACCCTTTGG 1306X60 12 10687002 AAGATCCCTGAATTTCCATCAGA 1307 X50 12 14689862AAAATACCTGCATACCCTCAGG 1308 RNA21 12 20320965 AACTTACATGAATTACTCTTAGG1309 X60 12 22372837 AACATTTCTCAATATCCCTTTGG 1310 X60 12 26607410CAGATAACTGAATAACCCTCTGT 1311 X30 12 28330833 TAAATACCTATAACCCACTGG 1312RNA32 12 28550781 GAGATACCTGAATATTCTTCCAA 1313 X60 12 32209572ATTATATCTGAATATCCTCTGG 1314 RNA41 12 45545836 ATGATACCCAATACCCCTCCAG1315 RNA41 12 47016633 CAGATACCTGAATCTCCTTCTTT 1316 X60 12 49068780CAGTTACCTGGATAACTCCCTGG 1317 X50 12 54882194 AAAATACTAGAATAACCAGCTTG1318 X60 12 61361291 AAGACTCAGGAATATCACTCAGG 1319 X60 12 66409389AAGATCCAGAAGAATCATCTGG 1320 RNA41 12 72361250 AAAACACCTGAATAAATCCCATG1321 X60 12 73122159 AACATCACTGAAGAACCCTGAAG 1322 X60 12 74390201AAAATACCTCATAGCCCTCTGC 1323 RNA41 12 77975069 AAAATAGCTTAATAATGCTCAGT1324 X60 12 78463857 AAGTTCCCTGAACACACCTCTGG 1325 X50 12 79756510ACTATACTGACTAACCCTCTAG 1326 RNA41 12 79913270 AGAATATCTTCATAACCTTCGGG1327 X60 12 81976639 AGAAAACCTGAATAACCTATGG 1328 RNA41 12 87296671AAGATATTCAAATAACCCTGCTG 1329 X60 12 98856708 GAGATACTGAATAACCTCTGGG 1330RNA41 12 105221258 AAGATAGCTGCTAACCCTGAGT 1331 RNA41 12 106415644AAAATAAATGAATAACCATCAGA 1332 X50 12 109568847 AAGGTACTAGAAAACCCTGTGG1333 RNA41 12 124304548 AAGACACTGCCAAACCCTCCGG 1334 RNA41 12 128916634AAGATACATGAATGGCCAACGCG 1335 X60 12 131173514 CAGAGACCTGCATAGCACTGAGG1336 X60 12 131502294 AAGATATCTGAAAATCCCTGCGC 1337 X50 13 20297900AAGAACACTGAGAAAACCCTCTGG 1338 DNA41 13 22964102 AAGAAACCTCCATATCCATCGTG1339 X60 13 24389118 AAGAAACCTCCATATCCATCGTG 1339 X60 13 25266108AAGGTACCTTAACAACATTTTGG 1340 X60 13 26448879 AAGGTGACTGAAGGACCCTTGGG1341 X60 13 26553083 ACGATAACTGAACAGCTCTCAAG 1342 X60 13 34458374AAGAGGGCTTAATAACCTTCAAG 1343 X60 13 39980150 AAGATAACTGGATAGCTCTATGA1344 X60 13 43747762 AAGATTCAGCAATACCCTCTGG 1345 RNA41 13 50325631CAAAAACCAGAAAACCCTCAGG 1346 RNA41 13 60126783 CATATACCAAAATAACCCATGGG1347 X60 13 61481200 AAAATACAAATAACCCTCAGA 1348 RNA32 13 66207288AAAATACCTGAATAAACCTGAAG 1349 X40 13 68049114 AAGATAACTTAATAGGCCTCCAC1350 X60 13 68754577 AAGATGGCTGAAAAAGCCTTTCG 1351 X60 13 74802880AAGCTGACTGAAGAGCCCTTGGG 894 X60 13 77274559 AAGTTACCAGATAACCCACAGG 1352RNA31 13 84432474 CAGATACCTGGTTAAACATCACG 1353 X60 13 86737129AAGATACATGAATGGCCAACAGG 1354 X50 13 92119936 AAGCTACCAGCATAGCCCACTAG1355 X60 13 109759900 AAGAAAGCTGGAAAACACTCCAG 1356 X60 13 111418659AAGATGCCTGAGTCTCCCCCGTG 1357 X60 13 112083112 AAGGAAACTGAATAACCCTCAGA1358 X40 13 113351476 CTGAGAACTGAATAAACCTCGAG 1359 X60 14 18485266AAGGTTACAGAATAAAACTCTGG 1360 X60 14 20357994 CAGGATACTGAATAACCCTCAGA1361 X60 14 21233079 CCATTACATGAATAACCTTCAGG 1362 X60 14 22348176AAGACACCTGGATAACCATCAGG 1363 X30 14 26263145 AAATTATGAAAAACCCTCAGG 1364RNA32 14 30878091 AAGATACTTGACAACTCTCTGA 1365 RNA41 14 31153973AAGATAGATGAATGACCCACTAC 1366 X60 14 35231283 AAGATACTTAAATGATCCTTGGC1367 X60 14 37392538 AATATACCTAAATAACTTACAGC 1368 X60 14 40710593AAGATAACTGGATAGCCATATGC 1369 X60 14 40860782 AACAAACATGAAAAACACTCAGC1370 X60 14 41931495 AAGATACCTAAATATATATCTGA 1371 X60 14 42141012AGGATGACTGAAAAATCCCCAGG 1372 X60 14 49513652 AAGATGACTGCTTAACCCCCAGT1373 X60 14 51112581 AAGATACCAAAATGTAACTCAGG 1374 X60 14 52885591AAGATAACTTAAAAACTCTAAGG 1375 X50 14 52900831 ACAGAACCTGAATAGCCCTAGGG1376 X60 14 53020328 AAGATGCCTGAACTAGTCCTTGGG 1377 DNA41 14 56099999AAAATAACTGAAAACCCTCAAC 1378 RNA41 14 59879739 AACATACATCCACAACCATCAGG1379 X60 14 64921531 AAGATACATGAATAAGCACCATG 1380 X50 14 66348379CATATACTTGAATAACCAACTGG 1381 X50 14 68160233 AAGATCTTAATAATCATCTGG 1382RNA32 14 72604917 AAGAAACCCGAGTCCCTCTGG 1383 RNA32 14 74660460AAGATATCTCATATCACTCAGG 1384 RNA41 14 74867740 ACAATACCTTGAATAACCCACAAG1385 DNA41 14 77627074 AAGATACATAAATAACCTCACAG 1386 X60 14 80214772AAGTAACAGAATAATCCTAAGG 1387 RNA41 14 81206628 GAATAACCTGAATAACCCCCAGA1388 X60 14 82199597 AAATTACCTAAATAACCCAAGGC 1389 X60 14 82935226AAGGTACCTGAATATTGTTCTGT 1390 X60 14 84727246 AAGACAGCTGGGTATCTCTCTGG1391 X60 14 87353900 CTGATAGTGAATAAGCCTCAGG 1392 RNA41 14 87564479ATGATAGTGAATAAGTCTCAGG 1393 RNA41 14 90316482 TAGATATTTGAATAACCATTTAG1394 X60 14 94330884 AAGATGCCCTGAGCAACCCTCCTG 1395 DNA41 14 100626641ATGATCTCTGAATGGCCTTCAGG 1396 X60 14 104781000 CGTGTACCTGAATGACCCACGGG1397 X60 14 106194663 TAGATACTGAGAAAGCCTCTGG 1398 RNA41 15 26338242AAGTTATCTAAAAACCCTCTAG 1399 RNA41 15 26400480 AAGATATCTGAAAAATTCTCAAA1400 X60 15 36010542 AAGTAAACTGAAAACCTTCTGG 1401 RNA41 15 37986317AATATAACTGAATAAAACTCTTT 1402 X60 15 38076935 TAGCTACATGAAAACCCTGGGG 1403RNA41 15 40609022 ATAATACTGAATAACCCTTTGA 1404 RNA41 15 43079813CTTTTACCTGAATACCCTCAGG 1405 RNA41 15 44348352 AAGATGACTGAAGAGCCCTTGGG1406 X50 15 49430075 ATGAGCCCAGAAGAACCCTCTTG 1407 X60 15 57756788GAGATACCTGAAAAATCCTGGCT 1408 X60 15 58607272 TGTATACCAGAATCACCCTAGGG1409 X60 15 60876750 CAGATACCTGGTAACTCTCAGG 1410 RNA31 15 61064287AAATTACCTGGACAACCTTCGCG 1411 X60 15 63004747 AAGATACATGAATAACCTTGTCA1412 X50 15 63156318 AAGATACATTAGAACCCTCTGT 1413 RNA41 15 69510497AAGATATATGAATGGCCCTGAAG 1414 X60 15 71127689 AAGATACACAAACAGCCCACAGG1415 X60 15 73446678 AACATACCTGAAAAAACCTCAGC 1416 X40 15 74253079AGGATTCCAGAAGAAGCCTGTGG 1417 X60 15 75470662 AAGAAACCTGGATTTACCTCTGT1418 X60 15 77101670 AGAATAGCTGAAGGACCCTGGGG 1419 X60 15 84875080AGGATGCCAGGGTAACCCACTGG 1420 X60 15 85440610 TAGGTACCTGAATGACCTCAGG 1421RNA31 15 85452623 AAGATACCTGACATACCCTCCCC 1422 X50 15 90002805GAGATACATGAATGACCCTTTCA 1423 X60 15 94867496 GTGATAGTGAATAACTCTCAGG 1424RNA41 15 96462615 AGGAAAGCTGAATAACTCTCTTA 1425 X60 15 96700894AAGAAACGCTAATAAACCTCAGG 1426 X50 15 99174006 AACATTCCACACTAACCATCAGG1427 X60 16 2950487 AAGATACGAGAGAAACGCCCGGG 1428 X60 16 7926633CAATTACCAGAATAACCCACAGA 1429 X60 16 11910562 AAGAGATTTGATAACCTTCTGG 1430RNA41 16 14200710 AAGATTCCTGATTCAGCCTCTGA 1431 X50 16 15502916AAGAAGCCAGACTAACACTGGGG 1432 X60 16 15969961 CAAATAAATGAATAATCATCAGG1433 X60 16 16048075 AAGAGGCCTGACTCTCCCTCCAG 1434 X60 16 17024068AAGATACTTGTATAACCTCAAGA 1435 X60 16 17094952 AACATACCTGAACAACCCTCAGG1436 X20 16 22614186 AAGATATTTATATACCCCTCCAG 1437 X60 16 24555817TAGTTACATGAAGAAACCTCTAG 1438 X60 16 25905845 AAGATACATGCGTGAAGCCTCTGG1439 DNA41 16 26382131 ATGGTACCTGTACAAACTTCAGG 1440 X60 16 27966927AAGGCACCTGAAAGCTCTCTGG 1441 RNA41 16 35693983 GAGATACCTGGAAAACCCCAGGC1442 X60 16 46657396 ATGTTAACAGAAGACCCCTCTGG 1443 X60 16 47691992TAGATAACTGGATACCCCACTGC 1444 X60 16 49245974 AAAATTCCTGTCCAGCCCTCTGG1445 X60 16 49984669 AGGATGCCTGAAAAAACCTCAAA 1446 X60 16 51935176AAGATACATGAGAACCCTTTGC 1447 RNA41 16 52525199 AAGATACATGAATAGCCCTCCAC1448 X40 17 5340518 TAAATACCTGAATAACCCATAGT 1449 X50 17 5857462TATATACATGTATAACCCACAGG 1450 X50 17 6194882 AAGATACATTCTTAGCCCTCAAG 1451X60 17 7932420 AAGAAACCTTGTGAACCCTCAGT 1452 X60 17 14407307AAGAAATGGATAGCCCTCTGG 1453 RNA32 17 21332996 AAAATAGCTTAAAAAGCCTGTGG1454 X60 17 27457685 AGGGTGCCCCAACAACCCTCGGG 1455 X60 17 28706042AAGAAATGTGGGTAAGCCTCAGG 1456 X60 17 29645114 ATCATATCTGAATAACTCTCAAC1457 X60 17 32458400 AACAAGCCTGTATAACCCTCATG 1458 X50 17 34969762GTGATACTGAATAAGTCTCAGG 1459 RNA41 17 37872545 AACATACATGAATAATCTTCAAA1460 X60 17 39300993 ATTATACCTCAATATCCCTCTGG 1461 X40 17 44931191AAGATACATGAAAAACACTGGCT 1462 X60 17 47455028 AACATACATGAATAATCTTCAAA1460 X60 17 47541148 AAGATACCTGTACAACACTATAC 1463 X60 17 54308512CAGATACCTCATTTACCCTGATG 1464 X60 17 65806019 CAGATACTTATAACCCTCTGG 1465RNA22 17 67990176 AAAATACTTGAAAAATCCCTCAGT 1466 DNA41 17 70972434AAGATCAAGAATAACTGTCAGG 1467 RNA41 17 81310828 AAGACACTTGTCTAGTCCTCTGG1468 X60 18 8119646 AAGAATCCGGAAAAACCCTATGC 1469 X60 18 10478571AAGACAGCTGGAGAACTCTCAGG 1470 X50 18 11443150 AAGAAAAATGAACAAACCTCAGA1471 X60 18 11651294 AAGTTACGTGAATAGGACTCAGT 1472 X60 18 13336259AAGATAAAGAACAACTCTCTGG 1473 RNA41 18 21942917 CAGATACACAGATAACCCCCAGG1474 X60 18 24883127 AAGAAACTTGAAAAATCCCAGG 1475 RNA41 18 35027674CAGATAACAGAAAAACCCTGTTG 1476 X60 18 36792668 AAGATACCCAGATCCCCCTCCAG1477 X60 18 36915438 TAGATATTCTGAATATACCTCTGG 1478 DNA41 18 39891882CAGATACATGAAATAATCCTCCAG 1479 DNA41 18 40348238 AGTCATCCTGAATAACCCTCATG1480 X60 18 47612935 GCCATACCAAATAACCCTCTGG 1481 RNA41 18 49237013AAGATACCTGGATAAGCAACTGC 1482 X50 18 49759577 AAGAAACCTATAGAACCCTGAGT1483 X60 18 52354807 AAGAAAACAGAATAAACCTCTAA 1484 X60 18 53136651AAGATACCTTATCAACCCTAAAG 1485 X50 18 54113085 CAGAAATATAAAAAACCCTCAGG1486 X60 18 55667190 AAGATTCAAGAAAGACCCTCTGC 1487 X60 18 56622476TAAATACTGAATAACCCTGTGA 1488 RNA41 18 58463120 GAGATCTCAGCATAACTCTCTGG1489 X60 18 66313025 AAGATACCTTATTTACCTATGG 1490 RNA41 18 68281088TTCATACCTAAATAACCCTTAGA 1491 X60 18 69322487 AAGATACCTGAATAACCTAAGAC1492 X50 18 74469327 AAGATACATAATAATCATCTGT 1493 RNA41 18 77464690AAGAGCCCTTGCTAACCATCTGG 1494 X60 19 11998269 AAAGTATCTGAATAACACTTCTG1495 X60 19 12455264 AGCCTACCTAAATAGCCCTAGGG 1496 X60 19 27693648CAGACACCTGAATGACCCCAGGC 1497 X60 19 28546228 CACTGACCTGAATCACCCCCAGG1498 X60 19 37641018 GAAATACTGAATAATCCTCAGG 1499 RNA31 19 38143521AAGACACAGAAAGATCCCTCTGG 1500 X60 19 42798163 AAGGTACCAGATGAGCCCTTGGG1501 X60 19 45644796 AGGATACCTCCCAAATCCTCTGG 1502 X60 19 46147476CAAATACTTGAAAAGCCCTCGAG 1503 X60 19 52963761 TAAATATGAATGACCCTCAGG 1504RNA32 19 53485175 CAGATATCTGAAGATCCCTCCAG 1505 X50 20 1471262ACAGAACCTGTATAACCCTCCAG 1506 X60 20 2545286 AAGAAATCTAGAATACCCCTAGGG1507 DNA41 20 6093655 AAGTTAAATGAATGACCCTGACCGG 1508 DNA42 20 7232430AAGATATTTGATCAACCCTTGGA 1509 X60 20 10350390 AGGTAACATCAATATCCCTCCGG1510 X60 20 12411793 AACGTACCTGAATAACTCATAAG 1511 X60 20 13449167AAGATACCAGCATAGTCCTCCTG 1512 X50 20 13559179 AAGATATCTGAAGAGCCTTCCCA1513 X60 20 18449496 AAGATACCTGAATTTACCATGAG 1514 X60 20 19443923AAGATACTTTATTAACTCTCAAG 1515 X50 20 22704701 TAGATATGTGAATTAACTTCTGG1516 X60 20 23658111 AAGATACCTGATCACACATCAGA 1517 X60 20 37569891CAGATACCTCAATAACCCTGATG 1518 X40 20 44858917 AAGATACTTGGATAATGCTTGGT1519 X60 20 45794486 AAGGTATCTGAATTACCCTCAAG 1520 X40 20 52287275CTGAGACCTGAGTAACTCTCATG 1521 X60 20 56292197 AAGAAAATGAATAACCCTACAG 1522RNA41 20 60198378 TAGATACATGAAAAACCCTTCAG 1523 X50 20 60509505TAGATGCTTAAATGACCCTCTGC 1524 X60 21 17021098 TAGGATCCTGGATAACCCTCCAG1525 X60 21 17566032 TTGATACTTGAATAACCATCTGA 1526 X50 21 17907925AAGAAACCAATAACCCTCAGG 1527 RNA12 21 19274949 AAGGAACCAGAATTACTCTCAGT1528 X60 21 24464330 CAGAAACCAGAATAACCTTCAGG 1529 X40 21 24537041AAAATTCCTGAATAGCTCTCTGG 1530 X40 21 26666529 TAGAGACATGAAAAACCCTCCAA 836X60 21 30710727 ATGATAGTGAATAACACTCATG 1531 RNA41 21 30757521AGGATACAGAATATCCATCTGG 1532 RNA41 21 31767749 AATCTACCTGAAAAGCCCTCTGG1533 X40 21 33249604 CTGATACCAGATAACCCTGAGG 1534 RNA41 21 45566319CAGATATCTGAATAACCCACCAG 1535 X40 21 46274280 AAGATACATGGATAATGTTCAGG1536 X50 21 46379614 AAGAAACATGAAGAAAACTCAGC 1537 X60 22 15415612AAGGTTACAGAATAAAACTCTGG 1360 X60 22 16210764 AAAATACATGGAAAATCCTCATG1538 X60 22 18016369 ATTATACCTGAATAAACCTGACT 1539 X60 22 22356999GATTTACCAGAAAAACCCTCTGG 1540 X50 22 22409053 GATTGACCAGAATAACCCTCTGG1541 X50 22 26261319 AAGATACATCGAATAACCCTTCTA 1542 DNA41 22 31956781AAGGCAACTGAAGATCCTTCAGG 1543 X60 22 48095647 AAGATATCTGAATTCAGCTCTTG1544 X60 X 3121703 AAGAAAACAGAATAAACCTCTCA 1545 X60 X 3288787AAGATAGCTGATGAAATATCAGG 1546 X60 X 6286346 ATGATAGTGAATAAGCCTCAGG 1547RNA31 X 15276107 AAGATTCATGTACAAACCTCATG 1548 X60 X 18754961AAGACACATGAAGAAAACTGAGG 1549 X60 X 19389428 AAGTTGACTGAAACCCTCTGG 1550RNA32 X 25123524 GAGATACCCTAGATAACCCTCAGA 1551 DNA41 X 33500389GTGATAACTGAATAACCCTGCTC 1552 X60 X 57574816 GAGATAACTGCATAGCCCTAGGT 1553X60 X 58085053 GAGACACCTGAACGACCTCAGG 1554 RNA41 X 58098997AAGACACCTGACTAACCCCAGGC 1555 X50 X 58100889 AAGACACCTGACTAACCCCAGGC 1555X50 X 58123415 TAGACACCTGAACAACCCCAGGC 1556 X60 X 58198267AAGACACCTGGCTGACCCCAGG 1557 RNA41 X 58199277 AAGACACCTGAGCAACCTCAGG 1558RNA31 X 58401983 AAGATATCTGAGCAACACTCTGT 1559 X50 X 63325513GAGATATGTGAATAAAACTCTGA 1560 X60 X 63786600 CAGAAGCTGAATGACCCTTTGG 1561RNA41 X 63945361 AAAATAACTGATAAATCCTCTGA 1562 X60 X 65679966AAGCTCCTGCATAGCCCACAGG 1563 RNA41 X 70140322 AACATACCTAAATATACCCTGG 1564RNA41 X 70754250 AAGCTGACTGAAGAGCCCTTGGG 894 X60 X 72198434TAGATACCTGACTTTCCCACTTG 1565 X60 X 73418101 AAGATTCCTGAAGCCTCCTCAGA 1566X60 X 73819519 CATATACTGAATAACCATCTGG 1567 RNA31 X 84823869AAGATATCTGAAATGCCCTAGAG 1568 X60 X 89194879 AAATAGCCTGAATAACCCTAGTG 1569X60 X 89316606 AACAAACATGAATAACACTAAGC 1570 X60 X 91030898AAGAAATCTGACAAACCTTTAGG 1571 X60 X 91177495 GTGATACTGAATAAGTCTCAGG 1459RNA41 X 99969578 AAGATCCAGAAGAACTCTCTGA 1572 RNA41 X 107292941TAGATACCTAAATAGCCCATGGG 1573 X50 X 108681302 AAGAAACCTCTATAAGCCTCTTA1574 X60 X 109943949 AATCTACTGAATAACTCTCAGG 1575 RNA31 X 111810669ATGAATACTGAATAACATTCAGG 1576 X60 X 113640910 AAGATACCAATAACCCTCAAA 1577RNA22 X 117399450 AATGTACCCGAACAACCCTCAGG 1578 X40 X 120228774CAGATAGCTGAAAGAACACTCAGG 1579 DNA41 X 125113165 AACATAACTGAATAACCATAAGG1580 X40 X 126715614 AATCAACCTGAATATCCATCAGT 1581 X60 X 130340267AAGATACCCATTCACCCTCTGG 1582 RNA31 X 130463899 AAAGAACCTAAAAAACCCTCTTG1583 X60 X 132229997 AAGATAGGGGAAAGCCCTCTGG 1584 RNA41 X 138021160AAGGCAAATGAATAACCCACAGC 1585 X60 X 139830410 AAAATACAAATAACCCTCAGT 1586RNA32 X 144953782 AAGATCCAGAAGAATTCTCTGG 1587 RNA41 X 150328565AGAATACCTGAATACCCCCAGA 1588 RNA41 X 154034869 CAAATACCTTAATCACCATGAGG1589 X60 Y 4605941 AAGAAATCTGACAAACCTTTAGG 1571 X60 Y 4641256GTGATACTGAATAAGTCTCAGG 1459 RNA41 Y 6194908 AAGATGCCAACATAAAGCTCAGG 1590X60 Y 13359292 TAAATACCTATAACCCTGAGG 1591 RNA32 Y 16565563AGCACACATGAATAACCCTAAGG 1592 X50 Y 22230617 AAGATCCCAGAATAACTACTGG 1593RNA41

TABLE 14 ANGPTL3 gRNA off-target site validation in human primaryhepatocytes Editing % Chromosome Location treated control Net Editing %1 62604219 61.93 0.39 61.54 1 2.28E+08 0.75 0.81 −0.06 1   2E+08 0.10.03 0.07 1 59422147 0.32 0.4 −0.08 1 76436411 0.3 0.26 0.04 1 199366620.26 0.2 0.06 1 1.8E+08 0.47 0.55 −0.08 2 1.7E+08 0.18 0.1 0.08 217555591 0.09 0.12 −0.03 2 87443936 0.27 0.26 0.01 2 2.26E+08 0.13 0.19−0.06 2 1.22E+08 0.48 0.62 −0.14 2 1.84E+08 0.26 0.36 −0.1 2 1.88E+080.26 0.22 0.04 3  8738135 0.28 0.23 0.05 3  539550 1.24 0.44 0.8 31.21E+08 0.26 0.27 −0.01 3 99319950 0.21 0.22 −0.01 4 1.73E+08 0.22 0.220 4 1.44E+08 0.48 0.54 −0.06 4 51965380 0.09 0.08 0.01 4 1.01E+08 0.090.08 0.01 4 26200297 0.14 0.11 0.03 4 74170265 0.54 0.3 0.24 5 1.14E+080.16 0.13 0.03 5 1.56E+08 0.47 0.59 −0.12 5  8704833 0 0 0 5 164048020.15 0.19 −0.04 6 1.13E+08 0.09 0.11 −0.02 6 45506487 0.3 0.36 −0.06 689636030 0.28 0.3 −0.02 6 18241159 0.08 0.1 −0.02 6 1.05E+08 0.45 0.390.06 6 84651006 0.15 0.11 0.04 6 1.26E+08 0.17 0.14 0.03 6 1.34E+08 0.230.29 −0.06 6 22811364 0.3 0.31 −0.01 6  6543792 0.08 0.15 −0.07 6 3993855 0.39 0.45 −0.06 6 51857715 0.19 0.29 −0.1 6 90026983 0.28 0.31−0.03 6 1.48E+08 0.15 0.17 −0.02 6 1.46E+08 0.32 0.35 −0.03 7 1.58E+080.5 0.45 0.05 7 1.05E+08 0.4 0.61 −0.21 7 18326887 0.03 0 0.03 781886792 0.23 0.47 −0.24 7 1.24E+08 0.62 0.44 0.18 7 1.14E+08 0.41 00.41 8 66947451 17.71 0.67 17.04 8 19687879 0.19 0.26 −0.07 8 764790790.44 0.44 0 8 1.09E+08 0.14 0.23 −0.09 8 88225407 0.44 0.57 −0.13 8 1.4E+08 0.29 0.34 −0.05 8 1.22E+08 0.42 0.57 −0.15 9 89929285 0.15 0.21−0.06 9 1.01E+08 0.41 0.49 −0.08 10 32570562 0.17 0.23 −0.06 11  23762220.05 0.11 −0.06 11 58705310 0.36 0.39 −0.03 11 80186841 0.24 0.24 0 1112052022 0.35 0.32 0.03 12 26607410 0.41 0.46 −0.05 12 64120945 0.260.27 −0.01 12 98856708 0.34 0.42 −0.08 12 71164415 0.42 0.37 0.05 1360126783 0.23 0.22 0.01 13 1.12E+08 0.59 0.76 −0.17 14 20357994 2.530.43 2.1 14 66348379 0.21 0.22 −0.01 14 81206628 0.28 0.28 0 14 223481760.74 0.68 0.06 14 76759328 0.07 0.04 0.03 15 94867496 0.32 0.42 −0.1 1560876750 0.3 0.27 0.03 15 43079813 100 99.93 0.07 16 17094952 7.87 1.576.3 16 52525199 0.83 0.69 0.14 17 39300993 0.08 0.11 −0.03 18 219429170.48 0.65 −0.17 18 47612935 0.24 0.3 −0.06 19 37641018 0.4 0.34 0.06 2060198378 0.4 0.35 0.05 21 45566319 0.26 0.35 −0.09 21 17566032 0.15 0.18−0.03 21 17021098 0.19 0.17 0.02 22 18016369 0.08 0.21 −0.13 X 1.07E+080.25 0.29 −0.04 X 1.17E+08 0.24 0.2 0.04 X 21471705 0.42 0.4 0.02 X73819519 0.25 0.3 −0.05 X  6286346 0.58 0.83 −0.25 X 1.14E+08 0.38 0.380

Modifications and/or truncations to either the spacer or tracr portionof the gRNA was assessed by alteration of the gRNA GA100 spacer(5′-AAGATACCTGAATAACCCTC-3′ (SEQ TD NO: 15)) (Table 15). Themodifications to the guide can serve to improve on-target editingefficiency and/or improve off-target editing efficiency. Additionally,four different ABE8.8 mRNAs were assessed (MA004, MA040, MA041, MA045;Table 23), in two different experiments (separated in the table below).Each of the gRNAs with an equivalent amount of in vitro transcribedABE8.8 mRNA (1:1 ratio by weight) were co-transfected into primary humanhepatocytes and primary cynomolgus hepatocytes at 5000, 2500, and 1250ng/RNA/mL and processed as described.

TABLE 15 gRNA and/or mRNA modifications improve gRNA specificityHuman Primary Hepatocytes Editing % at Position 6 (Dose, Replicate #)Protospacer  SEQ ID 5000, 5000, 2500, 2500, 1250, 1250, gRNA (5′-3′) NO:mRNA rep 1 rep 2 rep 1 rep 2 rep 1 rep 2 GA441 AAGATACCTG 15 MA004 ND47.29 52.04 49.69 41.45 42.72 AATAACCCTC GA442 AAGATACCTG 15 MA004 35.3432.48 35.2 35.78 31.54 27.9 AATAACCCTC GA472 AAGATACCTG 15 MA004 37.6536.08 37.98 32.46 28.85 32.1 AATAACCCTC GA473 AAGATACCTG 15 MA004 37.931.23 37.55 35.45 28.07 26.92 AATAACCCTC GA474 AAGATACCTG 15 MA004 30.5939.55 37.46 37.26 28.79 25.37 AATAACCCTC GA475 AGATACCTGA 248 MA00436.82 37.79 37.96 40.49 28.11 28.81 ATAACCCTC GA476 GATACCTGAAT 249MA004 37.2 37.7 42.27 40.86 31.97 31.48 AACCCTC GA477 ATACCTGAATA 250MA004 25.59 24.9 27.3 25.25 19.23 19.19 ACCCTC GA441 AAGATACCTG 15 MA04043.09 45.18 29.68 47.56 41.53 35.98 AATAACCCTC GA442 AAGATACCTG 15 MA04041.76 43.74 41.43 41.19 31.04 32.85 AATAACCCTC GA472 AAGATACCTG 15 MA04041.15 42.31 40.66 40.5 31.38 28.23 AATAACCCTC GA473 AAGATACCTG 15 MA04039.8 39 40.85 34.58 29.19 28.19 AATAACCCTC GA474 AAGATACCTG 15 MA04038.99 33.48 30.43 34.63 24.83 22.76 AATAACCCTC GA475 AGATACCTGA 248MA040 37.64 39.98 37.03 39.31 28.67 26.83 ATAACCCTC GA476 GATACCTGAAT249 MA040 41.08 39.14 37.69 38.01 28.8 27.26 AACCCTC GA477 ATACCTGAATA250 MA040 20.51 19.43 22.64 22.01 15.69 16.61 ACCCTC GA441 AAGATACCTG 15MA041 28.97 20.87 31.35 37.98 27.9 27.48 AATAACCCTC GA442 AAGATACCTG 15MA041 26.39 25.73 29.01 27.38 20.18 20.53 AATAACCCTC GA472 AAGATACCTG 15MA041 27.32 29.91 28.54 28.29 23.4 23.15 AATAACCCTC GA473 AAGATACCTG 15MA041 19.75 19.89 19.6 17.62 14.97 14.04 AATAACCCTC GA474 AAGATACCTG 15MA041 22.56 20.87 18.86 17.38 15.4 14.14 AATAACCCTC GA475 AGATACCTGA 248MA041 31.47 29.18 27.34 23.98 17.67 18.89 ATAACCCTC GA476 GATACCTGAAT249 MA041 31.01 33.84 29.37 28.62 19.77 21.75 AACCCTC GA477 ATACCTGAATA250 MA041 3.7 2.53 3.92 3.47 2.96 3.24 ACCCTCHuman Primary Hepatocytes  Editing % Verve Verveat Position 6 (Dose, Replicate #) gRNA Protospacer SEQ ID mRNA 5000,5000, 2500, 2500, 1250, 1250, ID (5′-3′) NO: ID rep 1 rep 2 rep 1 rep 2rep 1 rep 2 GA441 AAGATACCTG 15 MA004 33.26 34.23 42 36.89 34.57 31.5AATAACCCTC GA442 AAGATACCTG 15 MA004 27.1 35.52 36.03 32.59 28.21 32.14AATAACCCTC GA472 AAGATACCTG 15 MA004 32.58 22.71 27.15 38.51 29.44 32.3AATAACCCTC GA473 AAGATACCTG 15 MA004 28.63 27.84 18.2 36.76 31.84 29.58AATAACCCTC GA474 AAGATACCTG 15 MA004 25.26 22.74 26.59 35.61 28.96 26.24AATAACCCTC GA475 AGATACCTGA 248 MA004 28.61 34.79 30.52 35.29 29.1430.03 ATAACCCTC GA476 GATACCTGAAT 249 MA004 31.45 27.75 31.88 32.1126.99 30.15 AACCCTC GA477 ATACCTGAATA 250 MA004 15.07 13.4 15.01 18.7316.78 16.96 ACCCTC GA441 AAGATACCTG 15 MA045 20.49 21.6 26.32 26.1324.37 25.48 AATAACCCTC GA442 AAGATACCTG 15 MA045 25.65 17.51 25.36 18.7318.54 20.34 AATAACCCTC GA472 AAGATACCTG 15 MA045 20.87 15.13 19.67 19.2819.07 ND AATAACCCTC GA473 AAGATACCTG 15 MA045 14.84 14.05 20.14 15.9515.95 16.71 AATAACCCTC GA474 AAGATACCTG 15 MA045 15.81 16.04 17.08 20.1515.02 14.33 AATAACCCTC GA475 AGATACCTGA 248 MA045 24.33 31.08 25.5228.55 20.61 21.1 ATAACCCTC GA476 GATACCTGAAT 249 MA045 25.43 24.26 23.4526.05 21.47 23.35 AACCCTC GA477 ATACCTGAATA 250 MA045 5.06 2.84 3.074.65 3.75 3.01 ACCCTC

Off-target analysis for altered gRNA/mRNA combinations at the highestdose of 5,000 ng/RNA/mL was performed for two sites previouslyidentified that showed off-target editing (Table 16). The calculatedediting percentage across the protospacer was totaled, and the negativecontrol editing % was subtracted. This data shows that modifications tothe guide and/or to the ABE mRNA improve off-target editing efficiency.

TABLE 16 Modifications to the guide and/or to the ABE mRNA improveoff-target editing efficiency. gRNA mRNA OT Site A OT Site B HumanPrimary Hepatocytes-SUM total editing % (all positions) minus control,AVG of ~2 replicates GA441 MA004 9.785 1.605 MA040 7.17 0.85 MA041 −0.1−0.27 GA442 MA004 7.65 0.77 MA040 6.97 0.64 MA041 −0.005 −0.17 GA472MA004 0.36 −0.13 MA040 0.78 −0.185 MA041 −0.035 −0.205 GA473 MA004−0.145 −0.135 MA040 −0.1 −0.215 MA041 −0.085 −0.19 GA474 MA004 1.0750.03 MA040 1.545 0.39 MA041 −0.095 −0.14 GA475 MA004 2.79 0.39 MA0401.68 ND MA041 −0.13 −0.205 GA476 MA004 0.48 0.385 MA040 0.785 0.295MA041 −0.17 ND GA477 MA004 −0.13 −0.215 MA040 −0.48 −0.23 MA041 −0.225−0.14 Human Primary Hepatocytes-SUM total editing % (all positions) AVGof ~2 replicates GA441 MA004 6.575 2.345 MA045 1.36 0.865 GA442 MA0047.315 1.75 MA045 1.355 0.895 GA472 MA004 2.195 1.005 MA045 1.19 0.87GA473 MA004 1.47 1.025 MA045 1.495 0.98 GA474 MA004 2.145 1.055 MA0451.405 0.9 GA475 MA004 3.67 1.825 MA045 1.39 0.955 GA476 MA004 1.7751.675 MA045 1.505 0.84 GA477 MA004 1.31 0.975 MA045 1.135 0.83

Lipid nanoparticles containing ABE8.8 mRNA and a gRNA matching thehuman/cynomolgus 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID NO: 13) sequence(GA066) that targets the splice donor at the 5′ end of PCSK9 intron 1were formulated at a 1:1 ratio by weight. The LNPs were administered toprimary human hepatocytes and primary cynomolgus hepatocytes, usingvarious dilutions to assess for editing activity at differentconcentrations of test article. LNP containing Cas9 mRNA and a gRNAmatching a protospacer sequence in another gene, ANGPTL3, as a positivecontrol was used for these experiments. This Cas9 mRNA/gRNA combinationwere chosen because it had been previously observed to produce highlevels of genome-editing activity in primary human hepatocytes, primarycynomolgus hepatocytes, and cynomolgus liver in vivo. It was observedthe ABE8.8/GA066 LNP substantially outperformed the control LNP withrespect to editing, displaying much higher potency in both human andcynomolgus hepatocytes (FIG. 15 ).

Example 5. Pcsk9 Gene Editing in Mice

LNPs containing SpCas9 mRNA and mouse Pcsk9-targeting gRNA withdifferent tracr designs (GA052, GA053, GA054, and GA055) at a 1:1 weightratio were formulated. Wild-type C57BL/6 mice were dosed with 2 mg/kg ofthe LNP test article. Seven days after dosing, the mice were euthanizedand genomic DNA was harvested from mouse liver, and then assessed forediting of the target site with next-generation sequencing. GA052,GA054, and GA055 all outperformed GA053 that had a previously disclosedtracr design (FIG. 16 ).

Example 6. Pcsk9 Base Editing in Mice

LNPs containing ABE8.8 mRNA and a mouse Pcsk9-targeting gRNA at a 1:1weight ratio were formulated. This gRNA matches the5′-CCCATACCTTGGAGCAACGG-3′ (SEQ ID NO: 69) protospacer sequence, whichis the mouse ortholog of the human 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ IDNO: 13) sequence (matched by GA066, GA095, GA096, GA097 and GA346) thattargets the splice donor at the 5′ end of PCSK9 intron 1. Wild-typeC57BL/6 mice were dosed with 2 mg/kg of the LNP test article via thelateral tail vein or retro-orbital in a total volume of 10 ml/kg. Sevendays after dosing, the mice were euthanized and genomic DNA washarvested from mouse liver, and then assessed for base editing of thetarget splice site with next-generation sequencing. Approximately 60%editing of the target splice site was observed (mouse Pcsk9 intron 1splice donor) (FIG. 17 ), providing a preclinical proof of concept of abase-editing therapy knocking down PCSK9 in the liver in vivo.

In a subsequent study, LNPs containing ABE8.8 mRNA and a mousePcsk9-targeting gRNA at a 1:1 weight ratio were formulated, and dosed inwild-type C57BL/6 mice at doses ranging from 0-2.0 mg total RNA/kg. Thelow dose of 0.05 mg RNA/kg showed high editing (>45%), while saturationof base editing occurred around 0.25 mg RNA/kg dose (FIG. 18 ).

In an additional study, LNPs containing ABE8.8 mRNA and a mousePcsk9-targeting gRNA were formulated at different weight ratios of mRNAand gRNA ranging from 1:1-1:6 and 2:1-6:1. This gRNA matches the5′-CCCATACCTTGGAGCAACGG-3′ (SEQ ID NO: 69) protospacer sequence, whichis the mouse ortholog of the human 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ IDNO: 13) sequence (matched by GA066, GA095, GA096, GA097 and GA346) thattargets the splice donor at the 5′ end of PCSK9 intron 1. Wild-type micewere dosed with the LNP at 0.05 mg/kg total RNA dose. Additionally, twoindividual LNPs constituted with the same mRNA and two guides, GA255 andGA257, which matches the 5′-CCCATACCTTGGAGCAACGG-3′ (SEQ ID NO: 69)protospacer sequence but has modifications to the tracr to improvestability, were also injected into mice. Seven days after dosing, themice were euthanized and genomic DNA was harvested from mouse liver, andthen assessed for base editing of the target splice site withnext-generation sequencing (FIG. 19 ). Base editing was comparablebetween many of the ratios, with 1:6 and 6:1 (gRNA:mRNA) performing thepoorest. The guides with modifications to the tracr had increasedediting efficiency.

Example 7. Angptl3 Base Editing in Mice

LNPs containing ABE8.8 mRNA and mouse Angptl3-targeting gRNAs (GA258,GA259, GA260, GA349, GA353) at a 1:1 weight ratio were formulated.Wild-type C57BL/6 mice were dosed with LNP test article at 0.05 mg/kgtotal RNA dose. The mice were later euthanized and genomic DNA washarvested from mouse liver, and then assessed for base editing of thetarget splice site with next-generation sequencing (FIG. 20 ).

In Vivo Evaluation in Non-Human Primates (NHP) Example 8. Evaluation ofPCSK9 ABE

First, a 2-week study was performed to evaluate editing of the PCSK9gene in cynomolgus monkeys using specific gRNA and base editor nucleaseABE 8.8 through administration of LNPs (FIG. 21 ). The ABE8.8/GA066 LNPwas administered to cynomolgus monkeys via intravenous infusion at twodoses, 3 mg/kg (n=3 animals) and 1 mg/kg (n=2 animals) with the intentof producing high-level base editing of PCSK9 gene in the liver. Twoweeks after administration of test article, blood samples were collectedfor clinical chemistry assays and PCSK9 ELISA assay, and the animalsunderwent necropsy for collection of liver samples, 2 samples each fromeach of the 4 lobes of the liver (8 in total from each animal).

Editing analysis of the liver specimens was performed by next generationsequencing. At the 3 mg/kg dose, a mean of 55% editing of the adeninebase at the target splice site in the liver samples was observed; at the1 mg/kg dose, a mean of 24% editing of the adenine base at the targetsplice site in the liver samples was observed (FIG. 22 ). For PCSK9ELISA analysis, blood samples were collected from the animals on daysD-10, D-7 and D-5 pre-dose (average shown) as well as on D8 and D15post-dose. At 2 weeks after dosing there was a mean 76% reduction in theblood PCSK9 protein level compared to pre-dosing levels in the 3 mg/kggroup (FIG. 23 ), and a mean 32% reduction in the blood PCSK9 proteinlevel in the 1 mg/kg group (Table 17). Low density lipoproteincholesterol (LDL-C) was determined in serum samples taken pre-dose andon D8 and D15 using a standard clinical analyzer (Table 18). There was amean 57% reduction in the blood low-density lipoprotein cholesterol(LDL-C) level in the 3 mg/kg group (FIG. 24 ), and a mean 25% reductionin the blood LDL-C level in the 1 mg/kg group.

TABLE 17 Circulating PCSK9 protein levels decrease after PCSK9 gRNA/ABEediting Dose Concentration Reduction from PCSK9 (total RNA, Animal(ng/ml) basal on D15 Group mg/kg) ID Basal D8 D15 (%) 1 0 1001 432 387366 15 2 1 2001 324 285 227 30 2 1 2002 144 116 95 34 3 3 3001 254 16 2889 3 3 3002 170 34 31 82 3 3 3003 253 65 109 57

TABLE 18 LDL-C reduction after editing of the PCSK9 gene Dose LDL-Cconcentration Reduction from (total RNA, Animal (mg/dl) basal on D15Group mg/kg) ID Basal D8 D15 (%) 1 0 1001 76.7 70 57 26 2 1 2001 72.3 5554 25 2 1 2002 69.0 53 52 25 3 3 3001 57.0 23 17 70 3 3 3002 60.0 25 2263 3 3 3003 107.7 88 66 39

In a subsequent experiment, LNPs were delivered in monkeys viaintravenous infusion at a 1.0 mg/kg dose. For three monkeys thatunderwent necropsy at 2 weeks after LNP infusion, there was a mean 63%base editing frequency of the PCSK9 splice site adenine in the liver,with no bystander base editing observed elsewhere in the protospacer(FIG. 25 ); there was a mean indel frequency of 0.5%. The editing wasaccompanied by a mean 81% reduction in blood PCSK9 levels and a mean 65%reduction in blood LDL-C levels. For two monkeys that underwent necropsyat 24 hours after LNP infusion, there was a mean 48% editing frequency.

In a subsequent short-term dose-response study (0.5, 1.0, and 1.5 mg/kgdoses, three monkeys each, with necropsy at 2 weeks), all dosesachieved >50% mean base editing rates; PCSK9 editing and reductions inPCSK9 protein and LDL-C appeared to saturate at doses ≥1.0 mg/kg (FIG.26 ).

In these studies we assessed liver function tests and in some groupsnoted moderate rises in AST and ALT that largely resolved by the end ofthe first week and entirely resolved by 2 weeks after LNP infusion, withno adverse health events observed in any of the animals. In assayingbase editing in a wide variety of tissues, we found that the liver wasthe predominant site of editing, with much lower editing observed in thespleen and adrenal glands and minimal editing observed elsewhere (FIG.27 ).

TABLE 25 Numeric formats of tissue distribution of editing of the PCSK9exon 1 splice donor adenine base in three animals, as shown in FIG. 27.Tissue control NHP NHP #1 NHP #2 NHP #3 Liver 0.12 59.49 73.83 55.10Spleen 0.02 5.25 7.99 5.67 Adrenal gland (left) 0.02 1.81 7.49 1.79Adrenal gland (right) 0.10 1.54 0.22 2.18 Kidney (left) 0.10 0.63 0.890.24 Kidney (right) 0.06 0.64 0.27 0.32 Skin (injection site) 0.13 0.081.47 1.56 Mandibular lymph nodes 0.06 0.15 0.48 0.43 (left) Mandibularlymph nodes 0.06 0.18 0.17 0.96 (right) Mesenteric lymph nodes 0.04 0.120.15 0.12 Testis (left) 0.13 0.11 0.06 0.38 Testis (right) 0.07 0.160.09 0.38 Epididymis (left) 0.09 0.12 0.58 0.83 Epididymis (right) 0.800.25 0.79 0.52 Skeletal muscle 0.16 0.40 0.33 0.07 Duodenum 0.07 0.300.83 0.26 Jejunum 0.06 0.19 0.28 0.58 Colon 0.08 0.07 0.08 0.13 Lung(left) 0.01 0.16 0.22 0.18 Lung (right) 0.10 0.54 0.24 0.16 Brain 0.120.17 0.05 0.15

In an additional study, different tracr designs were assessed. LNPs weredelivered in NHPs via intravenous infusion at a 1.0 mg/kg total RNAdose, unless otherwise specified. Base editing of the liver wasassessed, and several guides outperformed GA066 with the literaturetracr (FIG. 28 ).

In the same NHP study, LNPs containing either: 1) Cas9 mRNA and a gRNAmatching 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ TD NO: 13) (GA097) protospacersequence; or 2) ABE8.8 mRNA and gRNA matching 5′-CCCGCACCTTGGCGCAGCGG-3′(SEQ ID NO: 13) (GA097) protospacer sequence, were deliveredintravenously into NHPs. Importantly, the method by which classicalCRISPR/Cas9 disrupts a gene by ultimately introducing an indel, isdifferent than base editing where a base mutation has occurred. Further,a target region that is highly amenable to CRISPR/Cas9 editing does notnecessarily mean base editing at that location will occur, and viceversa. This is highlighted in the NHP study, where the LNP containingSpCas9/gRNA resulted in low liver editing in the NHPs (<5%), while theLNP containing ABE8.8/gRNA had significantly higher editing close to 40%(FIG. 29 ) with a dose (1 mg/kg) lower than the SpCas9/gRNA dose (1.5mg/kg).

In another study, two LNPs were compared for base editing activity afterdelivery in NHPs via intravenous infusion at doses ranging from 0.5mg/kg-3.0 mg/kg. Both LNPs showed high efficacy, with the first LNP (LNP#1) outperforming the second LNP (LNP #2), at doses as low as 0.5 mg/kghaving greater than 50% average editing (FIG. 30 ). Similar to pastexperiments, the PCSK9 protein levels decreased significantly upon highlevel of base editing at the splice site, decreasing circulating PCSK9protein by 80-90% (FIG. 31 ).

In a long-term study (four animals, with liver biopsy at 2 weeks) LNPswere introduced via intravenous infusion at a higher dose of 3.0 mg/kgto assess drug tolerability and the durability of PCSK9 protein andLDL-C reductions resulting from PCSK9 editing. The liver biopsy samplesshowed a mean 66% base editing frequency (FIG. 32 ). Blood PCSK9 proteinlevels reached a trough by 1 week and have remained stable thereafterout to at least 6 months, settling at approximately 90% reductions (FIG.33 ). Blood LDL-C and lipoprotein(a) [Lp(a)] levels have similarlyachieved stable troughs persisting to 8 months, settling at ≈60% and≈35% reductions, respectively (FIG. 34 , FIG. 35 ).

There were transient, moderate rises in AST and ALT that entirelyresolved by 2 weeks following LNP infusion (FIG. 36 ), with no changesin any other liver function tests and no adverse health events observedto date (FIG. 37 ). Importantly, the persistence of PCSK9 and LDL-Creductions for 8 months with no late AST and ALT elevations demonstratesthat such a response, whatever its scale, does not adversely impact thetreatment's efficacy.

To evaluate ABE8.8/gRNA (protospacer matching GA346) LNP-mediatedoff-target editing in primary cyno hepatocytes and monkey liver samples,ONE-seq was performed with a synthetic cynomolgus genomic libraryselected by homology to the gRNA spacer sequence. This library wastreated with ABE8.8 protein and PCSK9 gRNA, and the top 48ONE-seq-nominated sites (FIG. 38 )—of which the PCSK9 target site wasthe very top site—was assessed with next-generation sequencing oftargeted PCR amplicons from LNP-treated versus untreated NHP samples(FIG. 39 ).

In LNP-treated primary cynomolgus hepatocytes, besides editing at thePCSK9 target site, there was off-target editing (mean <1%) evident atonly one site, designated C5, which has poor homology to the humangenome (FIG. 39 ). Assessing the same 48 sites in liver samples frommonkeys that were treated with a 1.0 mg/kg LNP dose (from theaforementioned dose-response study), low-level off-target editing (mean<1%) only at the C5 site (FIG. 40 ) was observed. No off-target editingwith a 0.5 mg/kg LNP dose and only low-level off-target editing with a1.5 mg/kg LNP dose (mean <1%) were detected.

Example 9. Evaluation of ANGPTL3 ABE

Similar to the NHP studies performed with the ABE/PCSK9 guide(s), LNPsformulated with ABE8.8 mRNA and a gRNA with the5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID NO: 14) (GA067) cynomolgus spacer wasadministered to cynomolgus monkeys via intravenous infusion at twodoses, 3 mg/kg (n=3 animals) and 1 mg/kg (n=2 animals) with the intentof producing high-level base editing of ANGPTL3 in the liver. Two weeksafter administration of test article, blood samples were collected forclinical chemistry assays and ANGPTL3 ELISA assay, and the animalsunderwent necropsy for collection of liver samples. Small segments fromthe center and periphery of each lobe of the liver were excised andflash frozen in liquid nitrogen and stored at −86 to −60° C. Splice-siteediting was analyzed by next generation sequencing, confirming that basechange occurred. At the highest dose (3 mg/kg), a mean of 61% editing ofthe adenine base was observed at the target splice site in the liver; atthe lowest dose (1 mg/kg), the editing observed was 28% at the targetsplice site in the liver (Table 19).

In addition, blood samples were collected pre-dose, on D7 and D15post-dose to determine serum ANGPTL3, and triglycerides. At two weeksafter dosing there was a mean 90% and 31% reduction in the blood ANGPTL3protein level compared to pre-dosing levels in the higher (3 mg/kg) andlower (1 mg/kg) dosing groups (Table 20).

TABLE 19 Liver Editing in cynomolgus monkeys Dose Animal Mean ± standardGroup (total RNA, mg/kg) ID deviation 1 0 1001  0.25 ± 0.06 4 1 400139.6 ± 2.7 4 1 4002 15.1 ± 3.9 5 3 5001 61.2 ± 3.2 5 3 5002 56.4 ± 5.2 53 5003 64.3 ± 2.6

TABLE 20 Knockdown of circulating cynomolgus monkey ANGPTL3 followinggene editing using ABE Dose Concentration Reduction from (total RNA,Animal (ng/ml) basal on D15 Group mg/kg) ID Basal D8 D15 (%) 1 0 1001 3947 41 −7 4 1 4001 56 15 31 45 4 1 4002 38 22 31 17 5 3 5001 31 1 1 98 53 5002 33 11 9 72 5 3 5003 70 1 1 99

Triglycerides were determined in serum samples taken pre-dose and on D8and D15 using a standard clinical analyzer. Serum triglyceride reductionin response to LNP-mediated ANGPTL3 base editing with ABE8.8 and GA067are summarized in Table 21. The levels of triglycerides were reduced by24% and 59% in response to doses of 1 mg/kg and 3 mg/kg base editorABE8.8 and GA067 targeting ANGPTL3.

TABLE 21 Triglyceride reduction in response to ANGPTL3 gene in liverTriglyceride Dose concentration Reduction from (total RNA, Animal(mg/dl) basal on D15 Group mg/kg) ID Basal D8 D15 (%) 1 0 1001 31.3 4341 −31 4 1 4001 71.0 46 49 31 4 1 4002 67.7 52 56 17 5 3 5001 59.3 23 2165 5 3 5002 41.3 33 25 40 5 3 5003 55.0 27 15 73

In another independent study, the same ABE8.8/GA067 LNP was administeredto cynomolgus monkeys via intravenous infusion at a dose of 3 mg/kg (n=3animals) with the intent of producing high-level base editing of ANGPTL3in the liver. Two weeks after administration of test article, bloodsamples were collected for clinical chemistry assays, and the animalsunderwent a liver biopsy for collection of liver samples. At the 3 mg/kgdose, a mean of 60% editing of the adenine base at the target splicesite was achieved in the liver biopsy samples. Concordantly, at 2 weeksafter dosing there was a mean 95% reduction in the blood ANGPTL3 proteinlevel compared to pre-dosing levels and there was a mean 64% reductionin the blood triglyceride level (FIG. 41 ). These results provide anadditional preclinical proof of concept of a base-editing therapyknocking down ANGPTL3 in the liver in vivo and effecting reductions inblood ANGPTL3 protein and triglyceride levels.

Example 10. Evaluation of Dual PCSK9 and ANGPTL3 Base Editing

To assess whether it was possible to affect simultaneous base editing ofPCSK9 and ANGPTL3 with a single test article, LNPs were formulatedcontaining a mix of three components: ABE8.8 mRNA, PCSK9-targeting gRNA(GA095), and ANGPTL3-targeting gRNA (GA098) at a 2:1:1 weight ratio.Primary human hepatocytes were incubated with various dilutions of theLNPs. Three days after incubation, genomic DNA was harvested from thehepatocytes, and then assessed for base editing of the target splicesite with next-generation sequencing. At the highest LNP concentrations,≈40% editing of the PCSK9 splice site (intron 1 splice donor) and ≈40%editing of the ANGPTL3 splice site (intron 6 splice donor) was observed(FIG. 42 ), demonstrating the feasibility of dual gene disruption inhuman hepatocytes with a single test article and providing a preclinicalproof of concept of a single base-editing therapy simultaneouslyknocking down PCSK9 and ANGPTL3, which would be predicted tosubstantially reduce both blood LDL cholesterol levels and bloodtriglyceride levels in human recipients. An advantage of this approachis that because the base editors do not require double strand breaks(DSBs) for editing, in contrast to standard CRISPR-Cas9, there issubstantially lower risk of chromosomal rearrangements or otherstructural changes inherent in the simultaneous targeting of twodifferent sites in the genome.

A follow-up NHP study was performed to address: 1) if a second dose ofadministered LNPs administered can cause editing of the target gene; 2)if a different target can be base edited. LNPs formulated with ABE8.8mRNA and either a gRNA targeting PCSK9 (GA346) or a gRNA targetingANGPTL3 (GA347) was administered to cynomolgus monkeys via intravenousinfusion at doses ranging from 0.5-2 mg/kg. Two weeks afteradministration of test article, biopsies were performed to assess forbase editing. After 30 days from the initiation of the study, theopposite LNP was administered. Following a second biopsy after anadditional 2 weeks, the gDNA was extracted, and base editing wasassessed using next generation sequencing. The findings from this studyshow that high level editing of both the PCSK9 and ANGPTL3 targets areachieved after the first and second doses of LNPs (FIG. 43 ). Bloodsamples were collected on both biopsy timepoints and show significantdecrease of around 90% in circulating PCSK9 and ANGPTL3 after subsequentdosing with 1 mg/kg LNPs (FIG. 44 ). In the same study an LNPencpasualting ABE8.8 mRNA, PCSK9 gRNA GA346 and ANGPTL3 gRNA GA347 at1:0.5:0.5 weight ratio was admnistred at 2 mg/kg total RNA dose, whichresulted in rubust synchronized PCSK9 and ANGPTL3 gene editing (FIG. 44, top and bottom data labelled GA346+GA347 (D1) - top and bottom panelsshow PSCK9 and ANGPTL3 editing). The data demonstrate that robustmulti-gene editing is possible with single 1-dose administration of ABEbase editor mRNA and two or more gRNAs targeting two or more genes ofinterest in mammals (and/or mammalian cell). FIG. 44 mirrorscorresponding knowckdown of ANGPTL3 (bottom) and PCSK9 (top) proteins.

In another study, NHPs were repeat dosed with LNPs (FIG. 45 ). NHPs weredosed with LNP #1 and LNP #2 formulated with ABE8.8 mRNA MA004 and agRNA targeting PCSK9 (GA097) via intravenous infusion at a total RNAdoses of 0.5 mg/kg. NHPs received an additional LNP dose on day 30 andon day 60. Liver biopsies from Day 14, Day 46, and Day75 were extractedfor adenosine base editing analysis. All gDNA was extracted, and baseediting was assessed using next generation sequencing. These resultsdemonstrate that repeat dosing of LNP containing ABE8.8 mRNA and PCSK9gRNA causes additive base editing in the liver, as editing efficiencywas near 30% after the first dose of LNP, while over 50% after the thirddose LNP. It was also observed that the magnitude of additive editingwas LNP-dependent.

Further, repeat dosing of LNPs reduced PCSK9 protein levels in NHPs(FIG. 46 ). The PCSK9 protein levels were monitored over 90 days in NHPsthat were repeat dosed with LNPs formulated with ABE8.8 mRNA MA004 and agRNA targeting PCSK9 (GA097). NHPs were dosed via intravenous infusionat a total RNA dose of 0.5 mg/kg at days 0, 30, and 60 (arrow isillustrated on graph to depict dosing). For description of analysis ofPCSK9 protein levels, see detailed methods section. Compared to basallevels, PCSK9 protein dropped by nearly 40% after the initial dose ofLNP, but upon additional doses, decreased even further.

Liver markers were assessed following repeat dosing of LNPs (FIG. 47 ).ALT, AST, total bilirubin, and creatine kinase levels were assessed upto 71 days in NHPs that were repeat dosed with LNPs formulated withABE8.8 mRNA MA004 and a gRNA targeting PCSK9 (GA097). NHPs were dosedvia intravenous infusion at a total RNA dose of 0.5 mg/kg at days 0, 30,and 60, which is shown as D1, D2 and D3 on graph, respectively. Liverbiopsy was performed at days 14 and 46 which is shown as B1 and B2 ongraph, respectively. Blood was collected at multiple timepoints, asdescribed on the graph. Although ALT and AST minimally rose upon dosing,this was a transient response that returned to normal after a period ofseveral days. Similarly, Creatine Kinase levels increased upon dosing,with the largest effect seen after the first dose, and returned to basallevels after a period of several days.

The liver enzymes, LDH, GLDH, GGT, and ALP, were assessed in NHPs thatwere repeat dosed with LNPs formulated with ABE8.8 mRNA MA004 and a gRNAtargeting PCSK9 (GA097) (FIG. 48 ). NHPs were dosed via intravenousinfusion at a total RNA doses of 0.5 mg/kg at days 0, 30, and 60. Bloodwas collected at multiple timepoints, as described on the graph.Although LDH and GLDH rose upon dosing, this was a transient responsethat returned to normal after a period of several days.

Further, long-term adenine base editing of ANGPTL3 was assessed innon-human primates (FIG. 49 ). Cynomolgus monkeys received anintravenous infusion of a 3 mg/kg dose of an LNP formulation with ABE8.8mRNA MA004 and ANGPTL3 gRNA GA067. Blood was collected at timepointsspecified and the graph, and ANGPTL3 protein levels (FIG. 49A) andtriglyceride levels (FIG. 49B) were analyzed. As compared to controls,both ANGPTL3 protein levels (96% reduction) and triglyceride levelssubstantially decreased upon base editing of the gene, and remain stablyreduced for more than 170 days.

Cytokine activation and immune response were assessed in NHPs receivingLNPs. In one set of studies, cynomolgus monkeys received intravenousinfusions of 0.5 mg/kg doses at three specified time points (FIG. 50Aand FIG. 50B) of an LNP formulation with ABE8.8 mRNA MA004 and PCSK9gRNA GA346. Blood was collected at timepoints specified and the graph,and IP-10 and MCP-1 were analyzed. These results demonstrated: 1) thatNHPs receiving LNPs had no evidence of cytokine activation nor immuneresponse, as compared to control; and 2) that repeat dosing of the sameLNP does not elicit cytokine activation nor an immune response.

In additional studies, IL-6, MCP-1, and SC5b-9 (FIG. 50C, FIG. 50D, andFIG. 50E, respectively) were analyzed at different time points fromblood collected from NHPs that received an intravenous infusion of 1.0mg/kg total RNA dose of LNP formulated with MA004 and PCSK9 gRNA GA346.This caused minimal cytokine/complement activation that returned tobaseline by/before 336 hours, as compared to control.

Example 11. Evaluation of SpCa9-Mediated On-Target Editing Efficiency

Gene editing of ANGPTL3 or PCSK9 in non-human primates was assessed.Cynomolgus monkeys received an intravenous infusion of a 1.5 mg/kg doseof an LNP formulation with SpCas9 mRNA MS004 and one gRNA targetingeither ANGPTL3 (GA261-GA263) or PCSK9 (GA266-GA271). Upon necropsy after2 weeks, two pieces from each liver lobe (8 pieces total) were isolatedand gDNA was extracted. Samples were processed as described in thedetailed methods section. Indel % was analyzed for each separate pieceand are graphed as individual points (FIG. 51 ). High editing efficiencywas observed in most NHP livers. LDL-C levels were measured (FIG. 52 ).All NHPs that received LNPs with SpCas9 mRNA/PCSK9 gRNA had at least 35%reduction in circulating LDL-C levels. Although more modest, LNPs withSpCas9 mRNA/ANGPTL3 gRNA had 10-25% reduction in circulating LDL-Clevels. Additionally, NHPs that received LNPs with SpCas9 mRNA/ANGPTL3gRNA had around 10-50% reduction in triglyceride levels (FIG. 53 ). NHPsthat received LNPs with SpCas9 mRNA/PCSK9 gRNA did not show asignificant reduction in triglyceride levels.

Cynomolgus primary hepatocytes were transfected at 2500, 1250, and 625ng/test article/mL with SpCas9 mRNA MS002 and a gRNA targeting PCSK9with modifications to the tracr. Genomic DNA was processed, sequenced,and analyzed as described in the detailed methods section. GA266 wastransfected independently twice to serve as two positive controls.Almost all transfections resulted in high editing efficiency compared topositive control, while GA405 had slightly lower editing efficiency.

TABLE 22 Modifications to guides retain on-target editing efficiency incynomolgus primary hepatocytes. Cynomolgus Primary Hepatocytes-% Editing(dose, replicate #) 2500, 2500, 1250, 1250, 625, 625, gRNA rep1 rep2rep1 rep2 rep1 rep2 GA266, #1 56.51 49.95 51.81 55.2 44.53 42.53 GA266,#2 50.34 48.3 43.56 46.2 39.75 36.59 GA395 43.91 49.53 41.48 44.35 33.8734.57 GA396 53.98 56.84 56.02 52.02 44.44 41.53 GA397 56.65 58.59 56.4758.68 40.72 46.28 GA398 59.85 63.97 56.45 55.21 39.09 44 GA399 58.9762.75 55.55 53.02 37.75 41.8 GA401 53.74 52.79 49.12 48.83 38.33 38.77GA402 45.58 55.34 44.46 47.93 35.05 40.31 GA403 52.62 55.84 50.5 47.1338.28 35.58 GA404 48.3 50.59 41.68 35.04 31.63 30.16 GA405 41.85 47.3335.84 35.88 26.2 26.36 GA406 57.17 51.45 45.11 43.68 37.58 40.31 GA40850.4 46.92 47.21 49.8 36.7 37.14

PACE-modifications to the gRNA have been previously demonstrated toreduce off-target editing efficiency. Human primary hepatocytes weretransfected at 2500, 1250, 500, and 250 ng/test article/mL with SpCas9mRNA MS002 and a gRNA targeting PCSK9 with modifications to the tracr.Genomic DNA was processed, sequenced, and analyzed as described in thedetailed methods section. GA156 was transfected to serve as a positivecontrol. GA248 and GA249 contain PACE-modifications to the gRNA thathave previously been demonstrated to decrease off-target editingefficiency. Indeed, although GA248 and GA249 had lower on-target editingcompared to the unmodified gRNA, GA156 (FIG. 54A), GA248 and GA249showed decreased off-target editing at an identified off-target site(FIG. 54B).

ABE mRNA sequences—MA004, MA019, MA020, and MA021—were evaluated fortheir GC content (FIG. 55A). In general, MA004 sequence has a higher GCcontent than MA019, MA020, MA021, and ABE8.8 m sequences. GC levels insequence MA004 are also elevated (above 60%) in certain regionsthroughout the ABE sequence such as the TadA domain, N-terminal linker,and C-terminal linker, as well as various sub-regions. For allsequences, GC levels below 60% are shaded in gray to provide contrast tothe elevated GC regions. GC content is calculated by determining therelative amount of G and C within a given 25 nucleotide stretch acrossthe entire mRNA sequence, i.e. for every 25 nucleotides, the sum of G'sand C's is divided by the total number of nucleotides. This comparisonshows that mRNA MA004 sequence has a distinct distribution andenrichment of G and C nucleotides relative to other mRNA sequences.

The region-specific GC characterization of sequence MA004 is furtherillustrated (FIG. 55 ). The graph in each row shows the GC content inevery 1,000-nucleotide stretch comprising the full ABE coding sequence(4,767 nt). The GC content is particularly high in the 5′ end of thesequence, which includes the TadA region and the N-terminal linker. TheGC content is also high various parts of the Cas9 nickase and in the 3′end of the ABE sequence; there the high GC % is centered around thelinker region instead of the NLS. Sub-regions with high GC content aredelineated by where GC reaches a threshold level of 60%. GC content ineach sub-region is calculated by determining the number of G and Cnucleotides divided by the total number of nucleotides for eachsub-region.

TABLE 26 GC comparison of ABE-encoding nucleotides, MA004, MA019, MA020,MA021, and ABE8.8m ABE sequence MA004 MA019 MA020 MA021 ABE8.8m AGC*Region-specific GC % Total sequence 63 54 47 54 55 11 TadA (nt: 1-501)70 58 54 60 54 14 N-terminal linker (nt: 502-597) 79 64 65 54 59 19 Cas9nickase (nt: 598-4698) 62 54 45 53 56 10 C-terminal linker (nt:4699-4710) 83 58 67 58 60 22 NLS (nt: 4711-4767) 63 53 49 60 48 11Sub-region GC % nt: 27-109 75 59 55 63 59 16 nt: 111-213 73 56 55 66 5715 nt: 250-385 71 60 57 62 57 12 nt: 389-475 70 59 56 54 53 15 nt:479-601 76 62 61 65 55 15 nt: 615-661 72 64 51 66 68 10 nt: 699-714 7563 63 56 69 12 nt: 735-829 74 62 54 61 61 15 nt: 1192-1345 67 58 51 5561 11 nt: 1363-1627 66 58 52 55 61 10 nt: 1661-1706 70 57 57 65 63 10nt: 1735-1880 66 54 52 57 60 10 nt: 1938-1999 71 60 55 63 60 12 nt:2023-2073 63 55 45 53 63 9 nt: 2184-2231 67 60 54 63 60 8 nt: 2542-260071 63 51 53 64 13 nt: 2719-2798 69 56 54 65 64 9 nt: 3294-3334 73 61 5461 66 13 nt: 3542-3584 72 67 58 72 67 6 nt: 3745-3864 67 54 53 57 62 11nt: 3977-4045 65 57 48 59 59 9 nt: 4207-4286 70 56 54 66 59 11 nt:4427-4517 65 60 51 53 55 10 nt: 4537-4569 73 58 48 64 61 15 nt:4583-4741 67 58 49 57 61 11 *AGC is the difference between the GCcontent of sequence MA004 CDS (cDNA sequence) and the average ofsequences MA019 CDS, MA020 CDS, MA021 CDS, and ABE8.8m.

The editing efficiency of these mRNA constructs were assessed in mice(FIG. 55B). These mRNAs, MA004, MAO 19, MA020, and MA021 code for thesame ABE protein sequence but have different nucleotide sequenceoptimizations. The only difference between these groups is the mRNAsequence; the mRNAs have the same base modifications, and each groupused the same LNP formulation and gRNA. Each group was administeredintravenously to mouse, and a low dose of 0.05 mg/kg was used in thisstudy to achieve sub-saturating levels of liver editing to resolveefficacy differences. At day 5 post-dose, gDNA was isolated and baseediting was assessed by next generation sequencing. These resultsdemonstrate that mRNA sequence changes can affect adenosine base editingperformance in vivo.

Additional Details of Methods Described

Plating, culturing, and transfection of primary hepatocytes. Primaryhuman liver hepatocytes (PHH) and primary cynomolgus liver hepatocytes(PCH) from BioIVT were cultured per the manufacturer's protocol.Briefly, primary human hepatocytes and primary cyno hepatocytes wereobtained as frozen aliquots from BioIVT. Four lots of primary humanhepatocytes, each derived from a de-identified individual donor, wereused for the experiments: STL (main donor) was used for all experiments,including screening experiments and off-target experiments; HLY, JLP,and TLY were used for off-target experiments. The HFG lot of primarycyno hepatocytes were used for experiments. Following the manufacturer'sinstructions, cells were thawed and rinsed prior to plating in 24-wellplates that had been coated with bovine collagen overnight, with adensity of approximate 350,000 cells/well in INVITROGRO hepatocytemedium supplemented with TORPEDO antibiotic mix (BioIVT). The cells werethawed and resuspended in hepatocyte thawing medium followed bycentrifugation at 100 g for 10 min at 4° C. The supernatant wasdiscarded, and the pelleted cells resuspended in hepatocyte platingmedium. Each vial contains approximately 5 million cells that were usedfor plating one 24-well plate. Plated cells were allowed to settle andadhere for 4-6 h in a tissue culture incubator at 37° C. under 5% CO₂atmosphere. After incubation, cells were checked for monolayerformation. The incubating media were then replaced with fresh hepatocytemaintenance media (complete INVITROGRO medium obtained from BioIVT, thecell line provider). The cells thus became ready for transfection. Eachof the gRNAs were co-transfected with an equivalent amount of in vitrotranscribed ABE8.8 mRNA (1:1 ratio by molecular weight) into primaryhuman hepatocytes via MessengerMax reagent (Lipofectamine), usingvarious dilutions to assess for editing activity at differentconcentrations of test article. MessengerMAX from Thermo Fisher is usedfor transfection. Solution A: desired amount of guide RNA is mixed with1:1 wt ratio of mRNA in OptiMEM. Solution B: MessengerMAX in OptiMEM.After mixing solutions A and B, the mixture was incubated at roomtemperature for 20 min. 60 μL of the incubated solution was addeddropwise to each cell wells. For protospacer sequences that were aperfect match to the corresponding cynomolgous monkey PCSK9 or ANGPTL3gene sequence, each gRNA was also co-transfected with an equivalentamount of ABE8.8 mRNA (1:1 ratio by molecular weight) into primarycynomolgous hepatocytes, and followed the same transfection protocol.The cells were then allowed to remain at 37° C. for 3 days. Cells wereharvested and prepared for genomic DNA extraction using either a ThermoKingfisher, or Qiagen DNEasy blood and Tissue Kit, per manufacturer'sinstructions.

Bioinformatic analysis. Targeted amplicon sequencing data were analyzedwith CRISPResso2 v2.0.31 in batch mode (CRISPRessoBatch). For Cas9experiments, the following parameters were set:“—quantification_window_center-3—quantification_window_size5—min_frequency_alleles_around_cut_to_plot0.1—max_rows_alleles_around_cut_to_plot 100”. For ABE experiments, thefollowing parameters were set: “—default_min_aln_score95—quantification_window_center-10—quantification_window_size10—base_editor_output-conversion_nuc_from A—conversion_nuc_toG—min_frequency_alleles_around_cut_to_plot0.1—max_rows_alleles_around_cut_to_plot 100”. For NHP experiments, anadditional parameter was set to exclude low quality reads:“—min_single_bp_quality 30”. Moreover, in all cases, the parameter“—max_paired_end_reads_overlap” was set to 2R-F+0.25*F, following FLASHrecommendations (http://ccb.jhu.edu/software/FLASH-1/), where R was theread length and F was the amplicon length.

Editing was quantified from the“Quantification_window_nucleotide_percentage_table.txt” output table asthe percentage of reads that supported any A-to-G/C/T substitution inthe main edited position (position 6 of the protospacer DNA sequence).Indels were quantified from the“Alleles_frequency_table_around_sgRNA_*.txt” output table as thepercentage of reads that supported insertions or deletions over a 5-bpwindow on either side of the nick site (at position −3 upstream of thePAM sequence), having excluded reads that supported deletions largerthan 30 bp. For candidate off-target sites, editing was quantified fromthe “Alleles_frequency_table_around_sgRNA_*.txt” output table as thepercentage of reads from alleles with an A—>G substitution in theediting window (positions 1-10 in the PAM distal side of theprotospacer), having excluded reads from alleles with deletions largerthan 30 bp.

Reverse transcription. The collected cells were processed with themiRNeasy Mini Kit (QIAGEN) according to the manufacturer's instructionsto isolate both large and small RNA species, with the other partharvested for genomic DNA to establish PCSK9 editing and thereby confirmbase editor activity in the cells. Reverse transcription was performedusing the iScript Reverse Transcription Supermix reagent according tothe manufacturer's instructions, with four different primer pairs usedfor PCR amplification of transcripts spanning exon 1 and exon 2, with orwithout any portions of intron 1. Paired-end reads of 250-bp lengthgenerated using an Illumina MiSeq System, as described above, weretrimmed for adapters using trimmomatic v0.39 with parameters“ILLUMINACLIP:NexteraPE-PE.fa:2:30:10:1:true LEADING:3 TRAILING:3SLIDINGWINDOW:4:15 MINLEN:36”. Reads were then merged with FLASHv1.2.1134 and aligned to the PCSK9 gene body with Bowtie2 v2.4.1 withparameters “—local-very-sensitive-local-k 1-np 0”. Gene annotations wereobtained from Ensembl v98(ftp://ftp.ensembl.org/pub/release-98/gtf/homo_sapiens/Homo_sapiens.GRCh38.98.gtf.gz).Alignments were filtered with samtools v1.10 and converted to BED formatwith the bedtools v2.25.0 bamtobed function. A minimum of 1000 mappedreads per sample were required and the end positions of mapped readswere tallied. Positions throughout PCSK9 intron 1 supported by a minimumof 10 reads in at least one treated sample are report.

ONE-seq analysis to predict candidate off-target sites. The design of aONE-seq library starts with the computational identification of sites ina reference genome that have sequence homology to the on-target. Forhuman ONE-seq libraries, the reference human genome (GRCh38, Ensemblv98, chromosomesftp://ftp.ensembl.org/pub/release-98/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.{1-22,X,Y,MT}.faandftp://ftp.ensembl.org/pub/release-98/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.nonchromosomal.fa),was searched for potential off-target sites with up to 6 mismatches tothe protospacer sequence above, and sites with up to 4 mismatches plusup to 2 DNA or RNA bulges, using Cas-Designer v1.2(http://www.rgenome.net/cas-designer/).

For the ONE-seq library pertaining to the cynomolgus monkey, thereference cynomolgus monkey genome (macFas5, Ensembl 98, chromosomesftp://ftp.ensembl.org/pub/release-98/fasta/Macaca_fascicularis/dna/Macaca_fascicularis.Macaca_fascicularis5.0.dna.chromos ome.{1-20,X,MT}.fa.gz andftp://ftp.ensembl.org/pub/release-98/fasta/Macaca_fascicularis/dna/Macaca_fascicularis.Macaca_fascicularis5.0.dna.nonchro mosomal.fa.gz) was searched using similar parameters.

Sites with up to 6 mismatches and no bulges are referred to using aX<number of mismatches> <number of bulges> code. As such, the on-targetsite is labelled as X00; a site with 1 mismatch to the on-target and nobulges is labelled as X10, and so on. Sites with DNA bulges are referredto with a similar nomenclature, DNA<number of mismatches> <number ofbulges>. As such, a site with 4 mismatches to the on-target and 2 DNAbulges is labelled as DNA42. The same nomenclature is used for RNAbulges, but these are coded as RNA<number of mismatches< >number ofbulges>.

The protospacer sequences identified were extended by 10 nucleotides(nt) on both sides with adjacent sequence from the respective referencegenome (these regions are herein referred to as the genomic context).These extended sequences were then padded by additional sequences up toa final length of approximately 200 nt, including 6 predefined constantregions of different nucleotide composition and sequence length; 2copies of a 14-nt site-specific barcode, one on each side of the centralprotospacer sequence; and 2 distinct 11-nt unique molecular identifiers(UMIs), one on each side of the central protospacer sequence. The UMIsare used to correct for bias from PCR amplification, and the barcodesallow for the unambiguous identification of each site during analysis.The barcodes are selected from an initial list of 668,420 barcodes,which contain neither a CC nor a GG in their sequences, and each barcodehas a Hamming distance of 2 from any other barcode. A custom Pythonscript was used for designing the final library.

The final oligonucleotide libraries are synthesized by a commercialvendor (Agilent Technologies). Each library is PCR-amplified andsubjected to 1.25×AMPure XP bead purification (Beckman Coulter). Afterincubation at 25° C. for 10 minutes in CutSmart buffer (New EnglandBiolabs), RNP comprising 769 nM recombinant ABE8.8-m protein and 1.54 μMgRNA is mixed with 100 ng of the purified library and incubated at 37°C. for 8 hours. The RNP dose is derived from an analysis documentingthat it is a super-saturating dose, ie, above the dose that achieves themaximum amount of on-target editing in the biochemical assay.

Proteinase K (New England Biolabs) is added to quench the reaction at37° C. for 45 minutes, followed by 2×AMPure XP bead purification. Thereaction is then serially incubated with EndoV (New England Biolabs) at37° C. for 30 minutes, Klenow Fragment (New England Biolabs) at 37° C.for 30 minutes, and NEBNext Ultra II End Prep Enzyme Mix (New EnglandBiolabs) at 20° C. for 30 minutes followed by 65° C. for 30 minutes,with 2×AMPure XP bead purification after each incubation. The reactionis ligated with an annealed adaptor oligonucleotide duplex at 20° C. for1 hour to facilitate PCR amplification of the cleaved library products,followed by 2×AMPure XP bead purification. Size selection of the ligatedreaction is performed on a PippinHT system (Sage Sciences) to isolateDNA of 150 to 200 bp on a 3% agarose gel cassette, followed by 2 roundsof PCR amplification to generate a barcoded library, which undergoespaired-end sequencing on an Illumina MiSeq System as described above.

Two cleavage products are obtained in a ONE-seq experiment. The PROTOside includes the part of the oligonucleotide upstream of the cleavageposition, whereas the PAM side includes part of the oligonucleotidedownstream of the cleavage position. In an ABE experiment, only thePROTO side is informative of editing activity (an A→G substitution);therefore, only this side is sequenced.

Paired-end reads were trimmed for sequencing adapters using trimmomaticv0.39 (Bolger et al., 2014) with custom Nextera adapters (PrefixPE/1:ACACTCTTTCCCTACACGACGCTCTTCCGATCT; PrefixPE/2:GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT; as specified in file) and parameters“ILLUMINACLIP:NEB_custom.fa:2:30:10:1:true LEADING:0 TRAILING:0SLIDINGWINDOW:4:30 MINLEN:36”. For experiments with lower sequencingquality (VOL014), these parameters were set to “ILLUMINACLIPNEB_custom.fa:2:30:10:1:true LEADING:2 TRAILING:0 SLIDINGWINDOW:30:30MINLEN:36”. Reads were then merged using FLASH v1.2.11 (Magoc andSalzberg, 2011) with parameters“—max-mismatch-density=0.25—max-overlap=160”. Merged reads were scannedfor the constant sequences, barcodes and protospacer sequences unique toeach site, and filtered to those with evidence of an A→G substitution inthe editing window (defined as the 1-10 most PAM-distal positions of theprotospacer). Duplicated reads were discarded.

For each site, the total number of edited reads was normalized to thetotal number of edited reads assigned to the on-target site, and thisratio defines the ONE-seq score for the site. Sites were ranked byONE-seq score, and those with a score equal to or larger than 0.001,were selected for validation. This implies the sponsor follows up onsites that have down to 1000-fold less editing activity in thebiochemical assay compared to editing of the on-target site. Thisthreshold is based on the premise that in cells, if there is 100%on-target editing, 1/1000-fold less editing activity would translate to<0.1% off-target editing, which falls below the lower limit of detectionof editing by NGS.

SureSelect. SureSelect panels were designed and purchased from AgilentTechnologies. Bases in raw FASTQ files with quality lower than 30 weremasked to Ns using seqtk v1.3-r106 (https://github.com/1h3/seqtk).Adapters were trimmed using the “Trimmer” script in Agilent's AGeNTv2.0.5 tool. Reads were aligned to the GRCh38 reference human genome(ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna)using BWA MEM v 0.7.17-r1188 with parameter “-C”. Aligned reads wereprocessed and duplicates were removed using the “LocatIt” script inAgilent's AGeNT v2.0.5 tool, with parameters “−1-R -IB -OB -U-1<Covered.bed>”. Nucleotide distributions at each position in theediting window (positions 1-10 in the PAM distal side of theprotospacer) were determined using perbase v0.6.3(https://github.com/sstadick/perbase), with parameters “base-depth-F3848”. Editing was quantified by summing the percentage of readssupporting an A→G substitution in the editing window.

RNA-seq for guide-independent off-target analysis. The RNA samples wereprocessed and sequenced by GENEWIZ; following rRNA depletion, librarieswere prepared and underwent 2×150-bp paired-end sequencing on anIllumina HiSeq System, with approximate 50 million reads per sample.RNA-seq variant calling for all samples was executed using GATK BestPractices. In short, reads were aligned using STAR to the GRCh38reference genome(ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz)with gencode v34(ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_34/gencode.v34.primary_assembly.annotation.gtf.gz).PCR duplicates were removed using GATK MarkDuplicates, followed byvariant identification using GATK HaplotypeCaller. Variants were thenfiltered by excluding those with QD (Quality of Depth)<2.0 and FS(Fisher strand—evidence of strand bias)>30. All GATK analyses wereperformed with gatk4 v4.1.8.1.

Variants obtained as above were further filtered by comparison withuntreated control samples as follows. (1) Nucleotide distributions ateach identified variant in treated cells each untreated control sampleand each treated sample using perbase v0.5.1(https://github.com/sstadick/perbase). (2) For all variants covered byat least 20 reads in both treated and untreated conditions, RNA editswere identified as those that had the reference allele (A or T) in atleast 95% of reads in all untreated control samples and the alternateallele (G or C) in at least one read in the treated sample. The abovesteps were executed with each of the ABE8.8-treated and SpCas9-treatedsamples.

Guide RNA synthesis. The guide RNAs shown in Table 1 were/aresynthesized under solid phase oligonucleotide synthesis and deprotectionconditions using controlled pore glass support and commerciallyavailable phosphoramidite monomers and oligonucleotide synthesisreagents (Methods in Molecular Biology, 1993, 20, 81-114; ACS Chem.Biol. 2015, 10, 1181-1187, incorporated herein by reference in itsentirety). The spacer section of the guide RNAs were converted to thecorresponding ribonucleotides except the first 1-3 nucleotides from the5′-end. The first 1-3 nucleotides from the 5′-end was/were converted tothe corresponding 2′-O-methylribonucleotide as outlined in Table 1. Thedeprotected guide RNAs were purified by HPLC and the integrity of eachguide RNA was confirmed by mass spectrometric analysis. The observedmass of each guide RNA was conformed to calculated mass.

mRNA production by in vitro transcription (IVT) The mRNA describedherein are produced by different methods well known in the art. One ofsuch methods is in vitro transcription (IVT) using T7 polymerase oradditional RNA polymerase variants. Typically, IVT of mRNA uses alinearized DNA template that comprises a T7 polymerase promoter andassociated regulatory sequences, mRNA coding sequence (CDS), 3′ and 5′untranslated regions (UTRs), poly A tail, and additional sequenceelements to enhance mRNA stability and in vivo performance. Prior toIVT, the DNA template is in the form of a plasmid, PCR product,synthetic DNA product, or any other double-stranded DNA construct;linearization of the DNA template, typically with a restrictiondigestion enzyme, is performed to promote run-off transcription. Atypical IVT reaction includes T7 polymerase, DNA template, RNaseinhibitor, cap analog, inorganic pyrophosphatase, and naturallyoccurring ribonucleotide triphosphates (NTPs) such as GTP, ATP, CTP,UTP, or substitutions of natural NTPs with modified NTPs such aspseudouridine, N1-methylpseudouridine, 5′methylcytidine,5-methoxyuridine, N6-methyladenosine, and N4-acetylcytidine. The capanalog can be added during transcription or supplemented after the IVTreaction using a capping enzyme; in both instances a 2′-O-methyl group,or additional 2′ chemical modification, is added to first initiatingnucleotide to produce a cap-1 form of mRNA. In some instances, poly Atail is added to the mRNA after the IVT reaction using an RNA ligaseenzyme. After IVT, in some cases DNase is added to the transcriptionmixture to remove DNA template; alternatively, residual DNA is removedwith chromatography, precipitation, or tangential flow filtration.Purification and concentration of mRNA is performed with methods such asion exchange chromatography, affinity chromatography, precipitation,ion-pairing reversed-phase chromatography, hydrogen bond chromatography,cellulose chromatography, reversed-phase chromatography, enzymaticreactions, size exclusion chromatography, and tangential flowfiltration. Similar IVT and purification process are used to producemRNA encoding luciferase, eGFP, SpCas9, Cas12b, CBE, and ABE; in allcases the DNA template, reaction conditions, and purification parametersare optimized for the specific gene of interest.

Lipid nanoparticle formulation and analysis. LNPs used were formulatedas previously described (Conway, A. et al. 2019 Mol. Ther. 27, 866-877;Villiger, L. et al 2021 Nat. Biomed. Eng. 5, 179-189) and generatedeither by (1) microfluidic mixing using the Precision NanosystemsNanoAssemblr system according to the manufacturer's protocol, with someoptimization for individual payloads or by (2) rapid inline mixing of asolution of lipid excipients in an organic solvent and an aqueoussolution of gRNA and mRNA. The lipid solution generally comprises of amixture of four formulation excipients namely: an amino lipid, amonomethoxypolyethylene glycol (or methoxypoluyethyle glycol) of averagemolecular weight 2000 Da conjugated to a lipid called PEG-Lipid,cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), mixedin a predetermined molar ratio in ethanol. LNP composition comprised40-65% of amino lipid, 2-20% DSPC, 1-5% PEG-Lipid, with the balancebeing cholesterol (all in mol %). The RNA aqueous solution contains a1:1 by weight mixture of desired mRNA and guide RNA (gRNA) unlessotherwise specified. For evaluating impact of mRNA to gRNA ratio,aqueous solution containing desired weight ratio of mRNA and gRNA wereprepared prior to the preparation of corresponding LNPs, for evaluation;for example, the preparation of LNP test article to evaluate 6:1, 3:1,2:1, 1:1, 1:2, 1:3 and 1:6 mRNA to gRNA ratio in mice (Example 6, FIG.19 ). In some other instances, the mRNA and two gRNAs were mixed in1:0.5:0.5 (mRNA:gRNA1:gRNA2) weight ratio to prepare LNPs containingdesired mRNA and two gRNAs in a single test article (Example 10, FIG. 43). The aqueous solution of desired mRNA to gRNA was then mixed with thelipid excipients in ethanol by microfluidic or by rapid inline mixing.All LNPs for reported NHP studies were prepared by following the inlinemixing protocols as described WO 2019/036028 A1, WO 2015/199952 A1, WO2017/004143, WO 2020/081938 A1 and WO 2020/061426 A2. The PEG-Lipid usedfor the preparation of the NHP LNP test articles was selected from WO2019/036028 A1, WO 2015/199952 A1. The LNP compositions and ionizableamino lipids were selected from the from Patent PublicationsWO/2017/004143A1, WO/2017/075531A1 and WO/2018/191719 A1. The ionizableamino lipids used for the preparation of LNP #1 and LNP #2 (Example 8,FIGS. 30 & FIG. 45 ) were selected from the publication WO 2018/191719A1. The resulting LNP formulations were subsequently dialyzed againsttest article buffer and filtered using a 0.2 μm sterile filter.

As an example, the LNPs used for cellular and NHP studies had an averagehydrodynamic diameter range of 55 to 65 nm, with a polydispersity indexof <0.2 as determined by dynamic light scattering and 85-98% total RNAencapsulation as measured by the Quant-iT Ribogreen Assay. The LNPparticle size (Z-Ave, hydrodynamic diameter), polydispersity index andtotal RNA encapsulation were measured as described in the literatureprior to administration.

As an example, the LNPs used for cellular and mouse studies had aparticle size of 55-120 nm (Z-Ave, hydrodynamic diameter), with apolydispersity index of <0.2 as determined by dynamic light scattering(Malvern NanoZS Zetasizer) and 85-100% total RNA encapsulation asmeasured by the Quant-iT Ribogreen Assay (Thermo Fisher).

Genomic DNA extraction. The whole mouse liver or 100-200 mg of monkeyliver was loaded into 2 mL lysing matrix tubes (MP Bio). Livers werelysed with 0.5 ml PBS for the mouse liver or 0.25 mL PBS for the monkeyliver, using the FastPrep-24 system (MP Bio) according to themanufacturer's protocol. Genomic DNA was isolated from approximately 20L of mouse or monkey liver lysate using a bead-based extraction kit,MagMAX-96 DNA Multi-Sample Kit (Thermo-Fisher Scientific) on theKingFisher Flex automated extraction instrument (Thermo-FisherScientific) according to the manufacturer's protocols. For monkey liverbiopsy samples, Qiagen DNEasy Blood & Tissue kit extraction was used toextract genomic DNA according to the manufacturer's instructions.Extracted genomic DNA was stored at 4° C. until further use or at −80°C. for long term storage.

PCSK9 protein levels quantified by ELISA. Blood samples were collectedand processed to plasma following blood draw. The plasma cynomolgusPCSK9 levels were determined by ELISA using the described ELISA;briefly, test samples or standards of purified cynomolgus monkey PCSK9diluted in assay diluent D (Biolegend, part #76384) were incubated withassay buffer A (Biolegend, part #78232) in a 96-well microplate coatedwith a monoclonal antibody specific for human PCSK9 (Biolegend, part#76157). After four washes with wash buffer (Biolegend, part #78233), apolyclonal antibody specific for human PCSK9 (Biolegend, part #76158)was incubated in individual wells. After four washes, avidin-HRP(Biolegend, part #77897) were next incubated in individual wells. Aftersix washes, substrate solution F (Biolegend, part #79132), whichcontains TMB, was used to develop the plate. Optical density wasdetermined using on a microplate reader set to 450 nm. Readings at 570nm were subtracted from the readings at 450 nm to correct for opticalimperfections in the plate. Blood samples were collected and processedto plasma following blood draw and stored at −86 to −60° C. untilanalysis.

ANGPTL3 protein levels quantified by ELISA. The plasma cynomolgusANGPTL3 levels were determined by ELISA; briefly, test samples orstandards of purified cynomolgus monkey ANGPTL3 diluted in calibratordiluent RD6Q (R&D, part #895128) were incubated with assay diluentRD1-76 (R&D, part #895812) in a 96-well microplate coated with amonoclonal antibody specific for human ANGPTL3 (R&D, part #893734).After four washes with wash buffer (R&D, part #895003), human ANGPTL3conjugate (R&D, part #893735) which contains a polyclonal antibodyspecific for human ANGPTL3 conjugated to horseradish peroxidase (HRP)were next incubated in individual wells. After four washes, TMBsubstrate solution (R&D, part #895000 and 895001) was used to developthe plate. Optical density was determined using on a microplate readerset to 450 nm. Readings at 540 nm were subtracted from the readings at450 nm to correct for optical imperfections in the plate.

Quantifying lipid levels. Reagent kits each analyte contain reagent,cholesterol, triglycerides and HDL-C are quantified using absorbancemeasurements of specific enzymatic reaction products. LDL-C isdetermined indirectly. Most of the circulating cholesterol is found inthree major lipoprotein fractions: very low-density lipoproteins (VLDL),LDL and HDL. [Total C]=[VLDL-C]+[LDL-C]+[HDL-C]. Thus the LDL-C can becalculated from measured values of total cholesterol, triglycerides andHDL-C according to the relationship: [LDL-C]=[total C]− [HDL-C]− [TG]/5,where [TG]/5 is an estimate of VLDL-cholesterol expressed. These resultsprovide a preclinical proof of concept of a base-editing therapyknocking down PCSK9 in the human liver in vivo and effecting reductionsin blood PCSK9 protein and LDL-C levels.

Direct measurement of triglyceride levels. A clinical analyzerinstrument is used to measure a ‘lipid panel’ in serum samples. Thisentails the direct measurement of cholesterol (total C), triglycerides(TG) and high-density lipoprotein cholesterol (HDL-C). A reagent kitspecific for triglycerides contains buffers, calibrators, blanks andcontrols. Using the provided reagents, serum samples from the study areanalyzed. Triglycerides are measured using a series of coupled enzymaticreactions. H₂O₂ is the end product of the last one and its absorbance at500 nm is used to quantify the analyte. The color intensity isproportional to triglyceride concentrations. All values are reported inmg/dL.

LNP treatment of mice. The mouse studies were approved by theInstitutional Animal Care and Use Committee of the Charles RiverAccelerator and Development Lab (CRADL), where the studies wereperformed. Female C57BL/6J mice were obtained from The JacksonLaboratory and used for experiments at 8-10 weeks of age, with randomassignment of animals to various experimental groups. LNP wereadministered to the mice via injection into the lateral tail vein and/orretro-orbital injection of the of the venous sinus (Lab Anim 2011;40(5): 155-160) at mg/kg doses that correspond to mg of total RNA peranimal weight (kilogram). The dose is calculated based on total RNA thatconstitute the amount of mRNA and gRNA, after formulating the LNP. Oneweek following treatment, the mice were euthanized unless otherwisestated, and liver samples were obtained on necropsy and processed withthe KingFisher Flex Purification System according to the manufacturer'sinstructions to isolate genomic DNA.

LNP treatment of NHPs. The NHP studies were approved by theInstitutional Animal Care and Use Committees of Envol Biomedical andAltasciences, respectively. NHPs studies were performed at EnvolBiomedical (study #VTP2001) and Altasciences (study #1388.02, 04, 05,09, and 11), with both studies using Macaca fascicularis) of Cambodianorigin. The animals were 2-3 years of age and 2-3 kilograms in weight atthe time of study initiation. All the animals were genotyped at thePCSK9 and/or ANGPTL3 editing site(s) to ensure that any animalsreceiving LNPs were homozygous for the protospacer DNA sequencesperfectly matching the gRNA sequence, and animals were randomly assignedto various experimental groups. The animals were premedicated with 1mg/kg dexamethasone, 0.5 mg/kg famotidine, and 5 mg/kg diphenhydramineprior to LNP administration unless otherwise stated. The LNP wereadministered using a temporary catheter inserted into a peripheral veinconnected to a primed infusion line, over the course of 1 hour (+/−5minutes). Dose formulations were administered at a volume of 6 ml/kg,unless otherwise specified, and dosed at mg/kg corresponding to mg oftotal RNA per animal weight (kilogram). The dose is calculated based ontotal RNA that constitute the amount of mRNA and gRNA, after formulatingthe LNP. The appropriate volume based on the weight of the animal wasdelivered using an infusion pump. Control animals receivedphosphate-buffered saline instead of LNP under the same infusionconditions. When there are two LNPs constituted from amino lipid 1 andamino lipid 2 used in the same NHP study, the test articles areidentified as LNP #1 and LNP #2 where mRNA and gRNA used for preparingthese LNPs are the same. If there is only one LNP composition used in astudy, the test article is identified as LNP

For blood chemistry samples, animals were fasted for at least 4 hoursbefore collection via peripheral venipuncture. NHP studies generallyfollowed collection on the following schedule: day −10, day −7, day −5,day 1 (6 hours after LNP infusion), day 2, day 3, day 5, day 8, and day15. In the long-term study, samples were also collected at day 21 andday 28 and have generally been collected every 2 weeks thereafter andanalyzed by the study site for LDL cholesterol, HDL cholesterol, totalcholesterol, triglycerides, AST, and ALT. For each analyte, the mean ofthe values at day −10, day −7, and day −5 were regarded as the baselinevalue. A portion of each blood sample was sent to the investigators forPCSK9 or ANGPTL3 protein measurement.

Analysis of cytokine levels. The serum cynomolgus IL,-6, MCP-1 and IP-10levels were determined by U-PLEX Biomarker Group 1 (NP) Assays (MesoScale Discovery, #K15068L-2) according to manufacturer's instruction.Briefly, U-PLEX (Meso Scale Discovery, #N05230) plate was incubatedovernight at 4° C. with linker-coupled capture antibodies (MCP-1antibody; Meso Scale Discovery, #C26UG-3, TL-6 antibody; Meso ScaleDiscovery, #C21TX-3, IP-10 antibody; Meso Scale Discovery, #C21UF-3).After 3 washes with PBS-T (PBS containing 0.050 Tween 20), test samplesor Calibrator standards (Calibrator 1; Meso Scale Discovery, #C0060-2,Calibrator 2: Meso Scale Discovery, #C0061-2) were incubated with assaydiluent 43 (Meso Scale Discovery, #R50AG-2), which contains serum,blockers and preservatives, at room temperature for an hour. After 3washes, SULFO-TAG conjugated detection antibodies for TL-6 (Meso ScaleDiscovery, #D26TX-3), MCP-1 (Meso Scale Discovery, #D26UG-3) and IP-10(Meso Scale Discovery, #D21UF-3) were incubated in individual wells atat room temperature for an hour. After 3 washes and adding Gold™ readbuffer B (Meso Scale Discovery, #R60AM-2) to each well, the plate wasanalyzed by MSD instrument (Meso Scale Discovery, #R31QQ-3.

Lengthy table referenced here US20230158174A1-20230525-T00001 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20230158174A1-20230525-T00002 Pleaserefer to the end of the specification for access instructions.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the disclosure described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment, anyportion of the embodiment, or in combination with any other embodimentsor any portion thereof.

As is set forth herein, it will be appreciated that the disclosurecomprises specific embodiments and examples of base editing systems toeffect a nucleobase alteration in a gene and methods of using same fortreatment of disease including compositions that comprise such baseediting systems, designs and modifications thereto; and specificexamples and embodiments describing the synthesis, manufacture, use, andefficacy of the foregoing individually and in combination including aspharmaceutical compositions for treating disease and for in vivo and invitro delivery of active agents to mammalian cells under describedconditions.

While specific examples and numerous embodiments have been provided toillustrate aspects and combinations of aspects of the foregoing, itshould be appreciated and understood that any aspect, or combinationthereof, of an exemplary or disclosed embodiment may be excludedtherefrom to constitute another embodiment without limitation and thatit is contemplated that any such embodiment can constitute a separateand independent claim. Similarly, it should be appreciated andunderstood that any aspect or combination of aspects of one or moreembodiments may also be included or combined with any aspect orcombination of aspects of one or more embodiments and that it iscontemplated herein that all such combinations thereof fall within thescope of this disclosure and can be presented as separate andindependent claims without limitation. Accordingly, it should beappreciated that any feature presented in one claim may be included inanother claim; any feature presented in one claim may be removed fromthe claim to constitute a claim without that feature; and any featurepresented in one claim may be combined with any feature in anotherclaim, each of which is contemplated herein. The following enumeratedclauses are further illustrative examples of aspects and combination ofaspects of the foregoing embodiments and examples:

Following is the first example of enumerated clauses:

-   1. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a PCSK9 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the PCSK9 gene in vivo when    administered to a mammalian subject,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.05 mg/kg, the base alteration occurs in at    least 35% of whole liver cells in the mammalian subject as measured    by next generation sequencing or Sanger sequencing.-   2. The composition of clause 1, wherein the mammalian subject is a    cynomolgus monkey,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.5 mg/kg, the base alteration occurs in at least    40% of whole liver cells in the cynomolgus monkey as measured by    next generation sequencing or Sanger sequencing.-   3. The composition of clause 1, wherein the mammalian subject is a    cynomolgus monkey,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 1 mg/kg, the base alteration occurs in at least    45% of whole liver cells in the cynomolgus monkey as measured by    next generation sequencing or Sanger sequencing.-   4. The composition of clause 1, wherein the mammalian subject is a    cynomolgus monkey,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 1.5 mg/kg, the base alteration occurs in at least    50% of whole liver cells in the cynomolgus monkey as measured by    next generation sequencing or Sanger sequencing.-   5. The composition of clause 1, wherein the mammalian subject is a    cynomolgus monkey,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 3 mg/kg, the base alteration occurs in at least    55% of whole liver cells in the cynomolgus monkey as measured by    next generation sequencing or Sanger sequencing.-   6. The composition of clause 1, wherein the mammalian subject is a    mouse,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.125 mg/kg, the base alteration occurs in at    least 40% of whole liver cells in the mouse as measured by next    generation sequencing or Sanger sequencing.-   7. The composition of clause 1, wherein the mammalian subject is a    mouse,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.5 mg/kg, the base alteration occurs in at least    45% of whole liver cells in the mouse as measured by next generation    sequencing or Sanger sequencing.-   8. The composition of clause 1, wherein the mammalian subject is a    mouse,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 2 mg/kg, the base alteration occurs in at least    50% of whole liver cells in the mouse as measured by next generation    sequencing or Sanger sequencing.-   9. The composition of clause 2, wherein the nucleobase alteration    results in a reduction of at least 50% in blood low-density    lipoprotein cholesterol (LDL-C) level in the subject as compared to    prior to the administration.-   10. The composition of any one of clauses 1-9, wherein the    protospacer is located in a splice site.-   11. The composition of any one of clauses 1-9, wherein the    protospacer complementary sequence is in the antisense strand of the    PCSK9 gene.-   12. The composition of any one of clauses 1-9, wherein the    protospacer complementary sequence is in the sense strand of the    PCSK9 gene.-   13. The composition of any one of clauses 1-12, wherein the base    alteration happens outside of the protospacer on the PCSK9 gene    (off-target sites),-   wherein the editing percentages of off-target sites set forth in    Table 11 are below or equal to the editing percentages set forth in    Table 11, respectively.-   14. The composition of any one of clauses 1-13, wherein the    deaminase is an adenine deaminase and wherein the nucleobase    alteration is a A•T to G•C alteration.-   15. The composition of any one of clauses 1-14, wherein the    programmable DNA binding domain comprises a nuclease inactive Cas9    or a Cas9 nickase.-   16. The composition of any one of clauses 1-15, wherein the    nucleobase alteration is at a splice site of the PCSK9 gene.-   17. The composition of clause 16, wherein the nucleobase alteration    is at a splice donor site of the PCSK9 gene.-   18. The composition of clause 17, wherein the splice donor site is    at 5′ end of PCSK9 intron 1 as referenced in SEQ ID NO: 5.-   19. The composition of clause 16, wherein the nucleobase alteration    is at a splice acceptor site of the PCSK9 gene.-   20. The composition of any one of clauses 1-19, wherein the    nucleobase alteration results in a frame shift, a premature stop    codon, an insertion or deletion in a transcript encoded by the PCSK9    gene.-   21. The composition of any one of clauses 1-20, wherein the    nucleobase alteration results in an aberrant transcript encoded by    the PCSK9 gene.-   22. The composition of any one of clauses 1-21, wherein the guide    RNA is chemically modified.-   23. The composition of clause 16, wherein the tracr sequence of the    guide RNA is chemically modified following the scheme depicted in    FIG. 7 .-   24. The composition of any one of clauses 1-22, wherein the spacer    sequence comprises a PCSK9 ABE guide RNA spacer sequence set forth    in Table 1.-   25. The composition of clause 24, wherein the guide RNA comprises    the PCSK9 ABE guide RNA sequence of GA096, GA097, GA343, GA346,    GA375-377, GA380-389, GA391, GA439 or GA440 as set forth in Table 1.-   26. The composition of any one of clauses 1-23, wherein the    protospacer sequence comprises a PCSK9 ABE protospacer sequence set    forth in Table 1.-   27. The composition of clause 26, wherein the protospacer comprises    the sequence 5′-CCCGCACCTTGGCGCAGCGG-3′ (SEQ ID No: 13) or    5′-CCGCACCTTGGCGCAGCGG-3′ (SEQ ID No: 247).-   28. The composition of any one of clauses 1-27, wherein the base    editor fusion protein comprises an amino acid sequence of SEQ ID No:    2137.-   29. The composition of any one of clauses 1-28, wherein the GC %    content of the mRNA sequence is greater than 50%.-   30. The composition of clause 29, wherein the GC % content of the    mRNA sequence is greater than 56%.-   31. The composition of clause 30, wherein the GC % content of the    mRNA sequence is greater than or equal to 63%.-   32. The composition of clause 29, wherein the mRNA comprises an    adenine tTNA deaminase (TadA) region, a Cas9 region and a nuclear    localization sequence (NLS) region.-   33. The composition of clause 32, wherein the mRNA further comprises    a first linker region which connects the TadA region and the Cas9    region, and a second linker region which connects the Cas9 region    and the NLS region.-   34. The composition of clause 32 or 33, wherein the GC % content of    the TadA region is greater than 60%.-   35. The composition of clause 32 or 33, wherein the GC % content of    the TadA region is greater than or equal to 70%.-   36. The composition of clause 32 or 33, wherein the GC % content of    the Cas9 region is greater than 56%.-   37. The composition of clause 32 or 33, wherein the GC % content of    the Cas9 region is greater than or equal to 62%.-   38. The composition of clause 32 or 33, wherein the GC % content of    the NLS region is greater than 54%.-   39. The composition of clause 32 or 33, wherein the GC % content of    the NLS region is greater than or equal to 63%.-   40. The composition of clause 33, wherein the GC % content of the    first linker region is greater than 65%.-   41. The composition of clause 33, wherein the GC % content of the    first linker region is greater than or equal to 79%.-   42. The composition of clause 33, wherein the GC % content of the    second linker region is greater than 67%.-   43. The composition of clause 33, wherein the GC % content of the    second linker region is greater than or equal to 83%.-   44. The composition of clause 33, wherein the GC % content of the    TadA region is greater than 60%, the GC % content of the Cas9 region    is greater than 56%, the GC % content of the NLS region is greater    than 54%, the GC % content of the first linker region is greater    than 65%, and the GC % content of the second linker region is    greater than 67%.-   45. The composition of clause 29, wherein the mRNA comprises a mRNA    sequence selected from Table 23.-   46. The composition of clause 45, wherein the mRNA comprises a mRNA    sequence of SEQ ID No: 2136.-   47. The composition of any one of clauses 29-46, wherein the mRNA    comprises a poly A tail.-   48. The composition of any one of clauses 1-47, further comprising a    lipid nanoparticle (LNP) enclosing (i).-   49. The composition of clause 48, wherein the LNP further encloses    (ii).-   50. The composition of clause 48, further comprising a second LNP    enclosing (ii).-   51. The composition of any one of clauses 1-44, wherein the ratio of    the guide RNA and the mRNA encoding the base editor fusion protein    is about 1:10 to about 10:1 by weight.-   52. The composition of clause 51, wherein the ratio of the guide RNA    and the mRNA encoding the base editor fusion protein is about 1:1,    1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, or 1:4 by weight.-   53. The composition of clause 51, wherein the ratio of the guide RNA    and the mRNA encoding the base editor fusion protein is about 1:1 by    weight.-   54. A pharmaceutical composition comprising the composition of any    one of the preceding clauses and a pharmaceutically acceptable    carrier or excipient.-   55. A method for treating or preventing a condition in a subject in    need thereof, the method comprising administering to the subject a    therapeutically effective amount of the composition of clause 1.-   56. The method of clause 55, wherein the administration is via    intravenous infusion.-   57. The method of clause 55 or 56, comprising sequential    administration of a LNP enclosing (i) and a LNP enclosing (ii).-   58. The method of clause 55 or 56, comprising concurrent    administration of the t LNP enclosing (i) and the LNP enclosing    (ii).-   59. The method of clause 57, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 1 day.-   60. The method of clause 57, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 2 days.-   61. The method of clause 57, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 3 days.-   62. The method of clause 57, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 4 days.-   63. The method of clause 57, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 5 days.-   64. The method of clause 57, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 6 days.-   65. The method of clause 57, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 7 days.-   66. The method of clause 55 or 56, comprising administering a single    dose of the LNP enclosing (i) and (ii).-   67. The method of clause 66, wherein the single dose of the LNP is    at about 0.3 to about 3 mg/kg.-   68. The method of clause 66 or 67, comprising administering a    treatment course of one or more treatments to the subject, wherein    each one of the one or more treatment comprises one or more of the    single doses of the LNP.-   69. The method of clause 68, comprising administering a treatment    course of two to ten treatments.-   70. The method of clause 68, comprising administering a treatment    course of two to five treatments.-   71. The method of clause 68, comprising administering a treatment    course of two treatments.-   72. The method of clause 68, comprising administering a treatment    course of three treatments.-   73. The method of clause 68, comprising administering a treatment    course of four treatments.-   74. The method of clause 68, comprising administering a treatment    course of five treatments.-   75. The method of any one of clauses 55-74, wherein the condition is    an atherosclerotic cardiovascular disease.-   76. The method of any one of clauses 55-74, wherein the condition is    an atherosclerotic vascular disease.-   77. The method of any one of clauses 55-74, wherein the subject is a    human.-   78. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a PCSK9 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the PCSK9 gene in vivo when    administered to a mammalian subject, and-   wherein the guide RNA comprises the PCSK9 ABE guide RNA sequences as    set forth in Table 1.-   79. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a PCSK9 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the PCSK9 gene in vivo when    administered to a mammalian subject, and-   wherein the mRNA comprises a sequence selected from Table 23.-   80. A method for treating or preventing an atherosclerotic    cardiovascular disease in a subject in need thereof, the method    comprising administering to the subject a therapeutically effective    amount of a first composition, comprising-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a PCSK9 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the PCSK9 gene in vivo when    administered to a mammalian subject,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.05 mg/kg, the base alteration occurs in at    least 35% of whole liver cells in the mammalian subject as measured    by next generation sequencing or Sanger sequencing; and-   a second composition, comprising-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a ANGPTL3 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the ANGPTL3 gene in vivo when    administered to a mammalian subject,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.5 mg/kg, the base alteration occurs in at least    35% of whole liver cells in the mammalian subject as measured by    next generation sequencing or Sanger sequencing.-   81. The method of clause 80, comprising sequential administration of    the first composition and the second composition.-   82. The method of clause 81, comprising administering one or more    doses of the first composition followed by one or more dose of the    second composition.-   83. The method of clause 82, comprising administering one or more    doses of the second composition followed by one or more dose of the    first composition.-   84. The method of clause 80, comprising concurrent administration of    the first composition and the second composition.-   85. The method of clause 84, comprising one or more doses of the    first composition and the second composition.-   86. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a APOC3 gene,-   wherein the guide RNA comprises the APOC3 ABE guide RNA sequence of    GA300-303 as set forth in Table 24.-   87. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a PCSK9 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the PCSK9 gene in vitro,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 2.5 mg/kg, the base alteration occurs in at least    35% of whole liver cells in the mammalian subject as measured by    next generation sequencing or Sanger sequencing.-   88. A composition for editing a PCSK9 gene comprising:-   (a) a mRNA encoding an adenine base editor protein having an editing    window, and-   (b) a guide RNA comprising a tracr sequence that serves as a binding    scaffold for the base editor protein and a spacer sequence that    serves to guide the base editor protein to a protospacer sequence on    the PCSK9 gene,-   wherein the spacer sequence is complimentary, at least in part, to a    splice site or an exon region of the PCSK9 gene.-   89. The composition for editing the PCSK9 gene of clause 88, wherein    when the base editor protein is operatively bound to the guide RNA    and the guide RNA is hybridized with the complementary strand to the    protospacer sequence on the PCSK9 gene, the editing window    encompasses the splice site of the PCSK9 gene.-   90. The composition for editing the PCSK9 gene of clause 88, wherein    when the base editor protein is operatively bound to the guide RNA    and the guide RNA is hybridized with the complementary strand to the    protospacer sequence on the PCSK9 gene, the editing window    encompasses a region of an intron of the PCSK9 gene.-   91. The composition for editing the PCSK9 gene of clause 88, wherein    when the base editor protein is operatively bound to the guide RNA    and the guide RNA is hybridized with the complementary strand to the    protospacer sequence on the PCSK9 gene, the editing window    encompasses a region of intron 1, intron 3 or intron 4 of the PCSK9    gene.-   92. The composition for editing the PCSK9 gene of clause 88, wherein    when the base editor protein is operatively bound to the guide RNA    and the guide RNA is hybridized with the complementary strand to the    protospacer sequence on the PCSK9 gene, the editing window    encompasses a region of intron 1 of the PCSK9 gene.-   93. The composition for editing the PCSK9 gene of clause 88, wherein    the spacer sequence has a 80-100% nucleotide sequence identity to a    spacer sequence selected from the group of guide RNA sequences    identified as GA066, GA073 and GA074.-   94. The composition for editing the PCSK9 gene of clause 88, wherein    the tracr sequence has a 80-100% nucleotide sequence identity to a    tracr sequence selected from the group of guide RNA sequences    identified as GA066, GA095, GA096, GA097, GA343, GA346, GA375,    GA376, GA377, GA380, GA381, GA382, GA383, GA384, GA385, GA386,    GA387, GA388, GA389, GA439, and GA440.-   95. The composition for editing the PCSK9 gene of clause 88, wherein    the mRNA has an 80-100%0 sequence identity to the mRNA sequences    identified as MA002, MA004, MA040, MA0041, or MA045.-   96. The composition for editing the PCSK9 gene of clause 88, wherein    the mRNA has one or more of the GC nucleotide region percentages set    forth in the following table:

Nucleotide region Average GC Nucleotide Content  27-213 67-73% 389-66167-71% 735-829 63-74% 4207-4286 67-70% 4537-4569 65-73% 4683-4741 62-67%

-   97. The composition for editing the PCSK9 gene of clause 88, wherein    the mRNA has one or more of the GC nucleotide region percentages set    forth in the following table:

Nucleotide region Average GC Nucleotide Content  27-213 At least 73%389-661 At least 71% 735-829 At least 74% 4207-4286 At least 70%4537-4569 At least 73% 4683-4741 At least 67%

-   98. The composition for editing the PCSK9 gene of clause 88, wherein    the mRNA and gRNA are encapsulated within a lipid nanoparticle.-   99. The composition for editing the PCSK9 gene of clause 88, wherein    the mRNA and gRNA are encapsulated within a lipid nanoparticle    having the following:    -   LNP composition (mol %):    -   40-65% iLipid    -   2-20% DSPC    -   1-5%0 PEG    -   Remaining mol % balance is cholesterol;    -   LNP Particle size: 55-120 nm Z average hydrodynamic diameter;        and    -   Polydispersity index of <0.2 as determined by dynamic light        scattering.-   100. The composition for editing the PCSK9 gene of clause 88,    wherein the mRNA and gRNA are encapsulated within the lipid    nanoparticle having an LNP particle size between 50-70 nm Z average    hydrodynamic diameter.-   101. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   102. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   103. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 30    percent.-   104. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   105. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   106. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 60    percent.-   107. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 70    percent.-   108. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   109. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   110. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 60    percent.-   111. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 70    percent.-   112. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when at administered to a group of    cynomolgus monkeys via intravenous infusion at a dose of    approximately 1.5 mg of the guide RNA and mRNA combined total weight    per kg of the cynomolgus monkey weight is capable of inducing    adenine base editing at the PCSK9 target splice site in the liver of    the cynomolgus monkeys with an average editing percentage of greater    than 80 percent.-   113. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   114. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   115. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 60    percent.-   116. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 70    percent.-   117. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the PCSK9 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 80    percent.-   118. The compositions for editing the PCSK9 gene of clauses 87-117,    wherein the percent editing is determined at 15 days after dosing    through analysis of dosed cynomolgus monkey liver either via liver    biopsy or necropsy of the monkey.-   119. The compositions for editing of the PCSK9 gene of clauses    87-117, wherein the percent editing is determined to be durably    maintained by periodic liver biopsy testing of the dosed cynomolgus    monkeys over a span of at least 168 days after dosing.-   120. The compositions for editing of the PCSK9 gene of clauses    87-117, wherein the percent editing is determined to be durably    maintained by periodic liver biopsy testing of the dosed cynomolgus    monkeys over a span of at least 300 days after dosing.-   121. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   122. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   123. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of monkey    weight is capable of reducing PCSK9 protein in the plasma of the    dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   124. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed monkeys on average of at least 60 percent as compared to    baseline.-   125. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   126. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   127. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   128. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   129. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of monkey weight    is capable of reducing PCSK9 protein in the plasma of the dosed    cynomolgus monkeys on average of at least 50 percent as compared to    baseline.-   130. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   131. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   132. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   133. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   134. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   135. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   136. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   137. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   138. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   139. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   140. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   141. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   142. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   143. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   144. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing PCSK9 protein in the plasma of    the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   145. The compositions for editing of the PCSK9 gene of clauses    121-144, wherein the reduction in plasma protein is determined at 15    days after dosing via blood sampling and analysis of the dosed    cynomolgus monkey.-   146. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of monkey    weight is capable of reducing LDL-C in the plasma of the dosed    monkeys on average of at least 20 percent as compared to baseline.-   147. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 25 percent as    compared to baseline.-   148. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 30 percent as    compared to baseline.-   149. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   150. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   151. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 45 percent as    compared to baseline.-   152. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 20 percent as    compared to baseline.-   153. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 25 percent as    compared to baseline.-   154. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 30 percent as    compared to baseline.-   155. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   156. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   157. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 45 percent as    compared to baseline.-   158. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   159. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 55 percent as    compared to baseline.-   160. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   161. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 20 percent as    compared to baseline.-   162. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 25 percent as    compared to baseline.-   163. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 30 percent as    compared to baseline.-   164. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   165. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   166. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 45 percent as    compared to baseline.-   167. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   168. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 55 percent as    compared to baseline.-   169. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   170. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 65 percent as    compared to baseline.-   171. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 20 percent as    compared to baseline.-   172. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 25 percent as    compared to baseline.-   173. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 30 percent as    compared to baseline.-   174. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   175. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   176. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 45 percent as    compared to baseline.-   177. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   178. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 55 percent as    compared to baseline.-   179. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   180. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing LDL-C in the plasma of the    dosed cynomolgus monkeys on average of at least 65 percent as    compared to baseline.-   181. The compositions for editing the PCSK9 gene of clauses 148-182,    wherein the reduction in LDL-C is determined at 15 days after dosing    via blood sampling and analysis of the dosed cynomolgus monkey.-   182. The compositions for editing the PCSK9 gene of clauses 148-182,    wherein the reduction in LDL-C is determined to be durably    maintained over a span of at least 168 days by periodic blood    sampling and analysis of the dosed cynomolgus monkey.-   183. The compositions for editing the PCSK9 gene of clauses 148-182,    wherein the reduction in LDL-C is determined to be durably    maintained over a span of at least 300 days by periodic blood    sampling and analysis of the dosed cynomolgus monkey.-   184. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) in the plasma of    the dosed cynomolgus monkeys on average of at least 10 percent as    compared to baseline.-   185. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 15    percent as compared to baseline.-   186. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 20    percent as compared to baseline.-   187. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 25    percent as compared to baseline.-   188. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 30    percent as compared to baseline.-   189. The composition for editing the PCSK9 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of approximately    35 percent as compared to baseline.-   190. The compositions for editing the PCSK9 gene of clauses 186-191,    wherein the reduction in lipoprotein(a) is determined at 15 days    after dosing via blood sampling and analysis of the dosed cynomolgus    monkey.-   191. The compositions for editing the PCSK9 gene of clauses 186-191,    wherein the reduction in lipoprotein(a) is determined to be durably    maintained over a span of at least 224 days by periodic blood    sampling and analysis of the dosed cynomolgus monkey.-   192. The compositions for editing the PCSK9 gene of clauses 186-191,    wherein the reduction in lipoprotein(a) is determined to be durably    maintained over a span of at least 300 days by periodic blood    sampling and analysis of the dosed cynomolgus monkey.-   193. The compositions for editing the PCSK9 gene of clauses 101-194,    wherein to the extent that the dosing of the cynomolgus monkeys    results in elevation of AST, ALT, or Cytokines, the elevations    resulting from the dosing of the composition are transient and    resolved back to approximately baseline levels within 3-15 days    after dosing.-   194. The composition for editing the PCSK9 gene of clauses 101-103,    121-126, 148-153, wherein the percent editing of PCSK9 is negligible    outside of the liver, spleen and adrenal glands tissues as    illustrated in FIG. 27 .-   195. The compositions for editing the PCSK9 gene of clauses 101-107,    121-132, 148-162, wherein repeat dosing results is additive with    respect to the editing percentage of PCSK9 editing percentage.-   196. The compositions for editing the PCSK9 gene of clause 195,    wherein the repeat dosing does not elicit cytokine activation nor an    immune response.-   197. The composition for editing the PCSK9 gene of clause 88,    wherein the spacer sequence has at least 80% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the PCSK9    gene, wherein an RNA nucleotide on the spacer sequence is in    correlation with a DNA nucleotide of the protospacer if it has the    same nucleotide as the DNA nucleotide in the same order and wherein    uracil and thymine bases are considered the same nucleotide for    purposes of determining correlation.-   198. The composition for editing the PCSK9 gene of clause 197,    wherein the spacer sequence has at least 85% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the PCSK9    gene.-   199. The composition for editing the PCSK9 gene of clause 197,    wherein the spacer sequence has at least 90% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the PCSK9    gene.-   200. The composition for editing the PCSK9 gene of clause 197,    wherein the spacer sequence has at least 95% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the PCSK9    gene.-   201. The composition for editing the PCSK9 gene of clause 197,    wherein the spacer sequence has at least 99% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the PCSK9    gene.-   202. The composition for editing the PCSK9 gene of clause 197,    wherein the spacer sequence has at least 100% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the PCSK9    gene.-   203. A method for treating or preventing an atherosclerotic    cardiovascular disease in a subject in need thereof, the method    comprising administering to the subject a therapeutically effective    amount of-   (a) a mRNA encoding an adenine base editor protein having an editing    window,-   (b) a first guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor protein and a spacer sequence    that serves to guide the base editor protein to a protospacer    sequence on a PCSK9 gene, wherein the spacer sequence is    complimentary, at least in part, to a splice site or an exon region    of the PCSK9 gene; and-   (c) a second guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor protein and a spacer sequence    that serves to guide the base editor protein to a protospacer    sequence on a ANGPTL3 gene, wherein the spacer sequence is    complimentary, at least in part, to a splice site or an exon region    of the ANGPTL3 gene.-   204. The method of clause 203, further comprising a first LNP    enclosing (a).-   205. The method of clause 204, wherein the first LNP encloses (b)    and (c).-   206. The method of clause 204, wherein the first LNP was    administered repeatedly.-   207. The method of clause 204, wherein the first LNP was    administered repeatedly at an interval of one to sixty days.-   208. The method of clause 204, wherein the first LNP was    administered repeatedly at an interval of seven days.-   209. The method of clause 204, wherein the first LNP further    encloses (b).-   210. The method of clause 209, further comprising a second LNP    enclosing (a) and (c).-   211. The method of clause 210, wherein the first LNP and the second    LNP are administered sequentially.-   212. The method of clause 211, wherein the first LNP and the second    LNP are administered sequentially at an interval of one day to 12    months.-   213. The method of clause 212, wherein the interval is one day.-   214. The method of clause 212, wherein the interval is five days.-   215. The method of clause 212, wherein the interval is ten days.-   216. The method of clause 212, wherein the interval is fifteen days.-   217. The method of clause 212, wherein the interval is twenty days.-   218. The method of clause 212, wherein the interval is twenty-five    days.-   219. The method of clause 212, wherein the interval is one month.-   220. The method of clause 212, wherein the interval is two months.-   221. The method of clause 212, wherein the interval is three months.-   222. The method of clause 212, wherein the interval is five months.-   223. The method of clause 212, wherein the interval is eight months.-   224. The method of clause 212, wherein the interval is ten months.-   225. The method of clause 212, wherein the interval is twelve    months.-   Following is the second example of enumerated clauses:-   1. A single guide RNA that comprises: (a) a spacer sequence, wherein    the spacer sequence comprises (i) one or more chemical    modification(s) and (ii) one or more unmodified nucleotide(s) at    select position(s), and (b) a tracr sequence having at least 70%    identity to SEQ ID NO: 61, wherein the tracr sequence serve as a    binding scaffold for a Type II Cas protein, and wherein the tracr    sequence comprises (i) one or more chemical modification(s) and (ii)    one or more unmodified nucleotide(s) at select position(s).-   2. The single guide RNA of clause 1, wherein the spacer sequence    hybridizes with a target polynucleotide sequence in a gene of    interest when contacted with the target polynucleotide sequence,    wherein the single guide RNA directs the Cas protein to effect    alteration in the gene when administered to a mammalian subject.-   3. The single guide RNA of clause 1 or 2, wherein the tracr sequence    comprises unmodified nucleotides at positions 2 to 7, 23 to 25, 27,    29, 31, 38, 39, 42 to 45 48, 49 and 62 as numbered in SEQ ID NO: 61    or a corresponding position thereof.-   4. The single guide RNA of clause 1 or 2, wherein the tracr sequence    comprises unmodified nucleotides at positions 2 to 7, 13, 23 to 25,    27, 29, 31, 38, 39, 42 to 45, 48, 49, 53 and 62 as numbered in SEQ    ID NO: 61 or a corresponding position thereof.-   5. The single guide RNA of clause 1 or 2, wherein the tracr sequence    comprises unmodified nucleotides at positions 2 to 7, 13, 23 to 25,    27, 29, 31, 38, 39, 42 to 45, 48, 49, 53, 61, 68, 70 and 11 as    numbered in SEQ ID NO: 61 or a corresponding position thereof.-   6. The single guide RNA of any one of clauses 1 to 3, wherein the    tracr sequence comprises modified nucleotides at positions 1, 8 to    22, 26, 28, 30, 32 to 37, 40, 41, 46, 47, 50 to 61, and 63 to 80 as    numbered in SEQ ID NO: 61 or a corresponding position thereof.-   7. The single guide RNA of any one of clauses 1 to 3, wherein the    tracr sequence comprises modified nucleotides at positions 1, 8 to    12, 14 to 22, 26, 28, 30, 32 to 37, 40, 41, 46, 47, 50 to 52, 54 to    61, and 63 to 80 as numbered in SEQ ID NO: 61 or a corresponding    position thereof.-   8. The single guide RNA of any one of clauses 1 to 7, wherein the    tracr sequence comprises modified nucleotides at positions 1, 8 to    12, 14 to 22, 26, 28, 30, 32 to 34, 37, 41, 46, 47, 50 to 52, 54 to    60, 62 to 67, 69 and 72 to 80 as numbered in SEQ ID NO: 61 or a    corresponding position thereof.-   9. The single guide RNA of any one of clauses 3 to 8, wherein more    than 60% of the nucleotides in the tracr sequence with SEQ ID NO: 61    are modified.-   10. The single guide RNA of clauses 9, wherein more than 70% of the    nucleotides in the tracr sequence with SEQ ID NO: 61 are modified.-   11. The single guide RNA of any one of clauses 1 to 10, wherein the    tracr sequence comprises unmodified nucleotides at positions 13, 49,    53 and 62 as numbered in SEQ ID NO: 61 or a corresponding position    thereof.-   12. The single guide RNA of any one of clauses 11, wherein the tracr    sequence comprises unmodified nucleotides at positions 49 and 62 as    numbered in SEQ ID NO: 61 or a corresponding position thereof.-   13. The single guide RNA of any one of clauses 1 to 10, wherein the    tracr sequence comprises unmodified nucleotides at positions 13, 40,    49, 53, 61, 68, 70 and 71 as numbered in SEQ ID NO: 61 or a    corresponding position thereof.-   14. The single guide RNA of any one of clause 13, wherein the tracr    sequence comprises unmodified nucleotides at positions 13, 49 and 53    as numbered in SEQ ID NO: 61 or a corresponding position thereof.-   15. The single guide RNA of any one of clauses 1 to 10, wherein the    tracr sequence comprises modified nucleotides at positions 1, 8, 21,    22, 26, 28, 30, 32 to 37, 40, 41, 46 and 47 as numbered in SEQ ID    NO: 61 or a corresponding position thereof.-   16. The single guide RNA of any one of clauses 1-15, wherein the    tracr sequence comprises at least 80% identity to SEQ ID NO: 61.-   17. The single guide RNA of any one of clauses 1-15, wherein the    tracr sequence comprises at least 90% identity to SEQ ID NO: 61.-   18. The single guide RNA of any one of clauses 1-17, wherein the    chemical modification comprises a 2′-OMe modification.-   19. The single guide RNA of any one of clauses 1-17, wherein the    chemical modification comprises a nebularin or a deoxynebularin.-   20. The single guide RNA of any one of clauses 1-17, wherein the    chemical modification comprises a phosphorothioate linkage.-   21. The single guide RNA of any one of clauses 1-20, wherein the    tracr sequence comprises SEQ ID No. 61, and wherein the single guide    RNA further comprises one or more phosphorothioate linkage at a 5′    end, at a 3′ end, at select internal positions or any combinations    thereof.-   22. The single guide RNA of clause 21, wherein the single guide RNA    further comprises two and no more than two contiguous    phosphorothioate linkages at the 5′ end, at the 3′ end or both.-   23. The single guide RNA of clause 21, wherein the single guide RNA    further comprises three contiguous phosphorothioate linkages at the    5′ end, at the 3′ end or both.-   24. The single guide RNA of any one of clauses 1-21, wherein the    single guide RNA comprises the sequence 5′- ususuNNN-3′ at the    3′end, wherein N independently indicates a unmodified    ribonucleotide, and wherein each u indicates 2′-O-methyluridine and    each s indicates phosphorothioate linkage.-   25. The single guide RNA of clauses 24, wherein each N is uridine.-   26. The single guide RNA of any one of clauses 1-21, wherein the    single guide RNA comprises the sequence 5′- ususuNNn-3′ at the    3′end, wherein each N independently indicates a unmodified    ribonucleotide, wherein the n indicates a modified nucleotide,    wherein each u indicates 2′-O-methyluridine and wherein each s    indicates phosphorothioate linkage.-   27. The single guide RNA of clauses 26, wherein each N is uridine    and the n is 2′-O-methyluridine.-   28. A single guide RNA that comprises (i) a spacer sequence and (ii)    a tracr sequence, wherein the spacer sequence hybridizes with a    target polynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene    when contacted with the target polynucleotide sequence, wherein the    tracr sequence binds a Type II Cas protein when contacted with the    Type II Cas protein, and wherein the single guide RNA comprises a    nebularine, a deoxynebularine, or a 2′-O-methylnebularine.-   29. A single guide RNA that comprises (i) a spacer sequence and (ii)    a tracr sequence, wherein the spacer sequence hybridizes with a    target polynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene    when contacted with the target polynucleotide sequence, wherein the    tracr sequence binds a Type II Cas protein when contacted with the    Type II Cas protein, and wherein the single guide RNA comprises two    and no more than two phosphorothioate linkages at a 5′ end or at a    3′ end.-   30. The single guide RNA of clause 29, wherein the single guide RNA    comprises two and no more than two phosphorothioate linkages at the    5′ end and at the 3′ end.-   31. The single guide RNA of clause 29, wherein the single guide RNA    comprises three phosphorothioate linkages at the 5′ end.-   32. The single guide RNA of clause 29, wherein the single guide RNA    comprises three phosphorothioate linkages at the 3′ end.-   33. The single guide RNA of clause 29, wherein the two    phosphorothioate linkages at the 5′ end are two contiguous    phosphorothioate linkages at the first two nucleotide positions of    the 5′ end.-   34. The single guide RNA of clause 29, wherein the two    phosphorothioate linkages at the 5′ end are within the first 3-10    nucleotides of the 5′ end.-   35. The single guide RNA of clause 29, wherein the two    phosphorothioate linkages at the 3′ end are two contiguous    phosphorothioate linkages at the last two nucleotide positions of    the 3′ end.-   36. The single guide RNA of clause 29, wherein the two    phosphorothioate linkages at the 3′ end are within the last 3-10    nucleotides of the 3′ end.-   37. The single guide RNA of clause 29 comprising the sequence    5′-UsUsUs-3′ at the 3′ end, wherein U indicates a uridine and s    indicates a phosphorothioate linkage.-   38. The single guide RNA of clause 29 comprising the sequence    5′-UUU-3′ at the 3′end.-   39. The single guide RNA of any one of clauses 1-27, wherein the    tracr sequence binds the Type II Cas protein with increased binding    affinity compared to a tracr sequence in an unmodified single guide    RNA.-   40. The single guide RNA of any one of clauses 28-38, wherein the    Type II Cas protein is a Cas9 protein.-   41. The single guide RNA of clause 40, wherein the Cas9 protein is a    Streptococcus pyogenes Cas9.-   42. A single guide RNA that comprises a guide RNA sequence selected    from Table 1, wherein a, u, g, and c indicate 2′-OMe modified    adenine, uridine, guanine, and cytidine, wherein s indicates a    phosphorothioate linkage, wherein X indicates a nebularine, wherein    x indicates a 2′-O-methylnebularine and wherein dX indicates a    2′-deoxynebularine.-   43. A pharmaceutical composition for gene modification comprising    the single guide RNA of any one of clauses 1-42 and a Type II Cas    protein or a nucleic acid sequence encoding the Type II Cas protein.-   44. The pharmaceutical composition of clause 43 further comprising a    vector that comprises the nucleic acid sequence encoding the Type II    Cas protein.-   45. The pharmaceutical composition of clause 43 or 44, wherein the    Type II Cas protein is a Cas9.-   46. The pharmaceutical composition of any one of clauses 43-45    further comprising a pharmaceutically acceptable carrier.-   47. A lipid nanoparticle comprising the pharmaceutical composition    of any one of clauses 43-46.-   48. A method for modifying a target polynucleotide sequence in a    cell comprising introducing into the cell the pharmaceutical    composition of any one of clauses 43-46, wherein the single guide    RNA directs the Type II Cas protein to effect a modification in the    target polynucleotide sequence in the cell.-   49. The method of clause 48, wherein the target polynucleotide    sequence is in a PCSK9 gene.-   50. The method of clause 49, wherein the modification reduces    expression of functional PCSK9 protein encoded by the PCSK9 gene in    the cell.-   51. The method of clause 50, wherein the target polynucleotide    sequence is in an ANGPTL3 gene.-   52. The method of clause 51 wherein the modification reduces    expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene    in the cell.-   53. The method of any one of clauses 48-52, wherein the introduction    is performed via a lipid nanoparticle that comprises the    composition.-   54. A method for treating or preventing a condition in a subject in    need thereof, the method comprising administering to the subject the    pharmaceutical composition of any one of clauses 43-46 or the lipid    nanoparticle of clause 47, wherein the single guide RNA directs the    Type II Cas protein to effect a modification in a target    polynucleotide sequence in a cell of the subject, thereby treating    or preventing the condition.-   55. The method of clause 54, wherein the target polynucleotide    sequence is in a PCSK9 gene.-   56. The method of clause 54, wherein the target polynucleotide    sequence is in an ANGPTL3 gene.-   57. The method of clause 55, wherein the modification reduces    expression of functional PCSK9 protein encoded by the PCSK9 gene in    the subject.-   58. The method of clause 56, wherein the modification reduces    expression of functional ANGPTL3 protein encoded by the ANGPTL3 gene    in the subject.-   59. The method of any one of clauses 48-58 wherein the condition is    an atherosclerotic vascular disease.-   60. The method of any one of clauses 48-58, wherein the condition is    an atherosclerotic vascular disease, hypertriglyceridemia, or    diabetes.-   61. The method of any one of clauses 48-60, wherein the subject    exhibits a reduced blood LDL cholesterol level, and/or a reduced    blood triglycerides level as compared to before the administration.-   Following is the third example of enumerated clauses:-   1. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a ANGPTL3 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the ANGPTL3 gene in vivo when    administered to a mammalian subject,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.5 mg/kg, the base alteration occurs in at least    35% of whole liver cells in the mammalian subject as measured by    next generation sequencing or Sanger sequencing.-   2. The composition of clause 1, wherein the mammalian subject is a    cynomolgus monkey,-   wherein when the guide RNA and the mRNA is administered at a total    amount of about 1 mg/kg, the base alteration occurs in at least 35%    of whole liver cells in the cynomolgus monkey as measured by next    generation sequencing or Sanger sequencing.-   3. The composition of clause 1, wherein the mammalian subject is a    cynomolgus monkey,-   wherein when the guide RNA and the mRNA is administered at a total    amount of about 3 mg/kg, the base alteration occurs in at least 50%    of whole liver cells in the cynomolgus monkey as measured by next    generation sequencing or Sanger sequencing.-   4. The composition of clause 2, wherein the nucleobase alteration    results in a reduction of at least 20% in blood triglyceride level    in the cynomolgus monkey as compared to prior to the administration.-   5. The composition of clause 3, wherein the nucleobase alteration    results in a reduction of at least 50% in blood triglyceride level    in the cynomolgus monkey as compared to prior to the administration.-   6. The composition of any one of clauses 1-5, wherein the    protospacer is located in a splice site.-   7. The composition of any one of clauses 1-5, wherein the    protospacer complementary sequence is in the antisense strand of the    ANGPTL3 gene.-   8. The composition of any one of clauses 1-5, wherein the    protospacer complementary sequence is in the sense strand of the    ANGPTL3 gene.-   9. The composition of any one of clauses 1-8, wherein the base    alteration happens outside of the protospacer on the ANGPTL3 gene    (off-target sites),-   wherein the editing percentages of off-target sites set forth in    Table 14 are below or equal to the editing percentages set forth in    Table 14, respectively.-   10. The composition of any one of clauses 1-9, wherein the deaminase    is an adenine deaminase and wherein the nucleobase alteration is a    A•T to G•C alteration.-   11. The composition of any one of clauses 1-10, wherein the    programmable DNA binding domain comprises a nuclease inactive Cas9    or a Cas9 nickase.-   12. The composition of any one of clauses 1-11, wherein the    nucleobase alteration is at a splice site of the ANGPTL3 gene.-   13. The composition of clause 12, wherein the nucleobase alteration    is at a splice donor site of the ANGPTL3 gene.-   14. The composition of clause 13, wherein the splice donor site is    at 5′ end of ANGPTL3 intron 6 as referenced in SEQ ID NO: 7.-   15. The composition of clause 12, wherein the nucleobase alteration    is at a splice acceptor site of the ANGPTL3 gene.-   16. The composition of any one of clauses 1-15, wherein the    nucleobase alteration results in a frame shift, a premature stop    codon, an insertion or deletion in a transcript encoded by the    ANGPTL3 gene.-   17. The composition of any one of clauses 1-16, wherein the    nucleobase alteration results in an aberrant transcript encoded by    the ANGPTL3 gene.-   18. The composition of any one of clauses 1-17, wherein the guide    RNA is chemically modified.-   19. The composition of clause 16, wherein the tracr sequence of the    guide RNA is chemically modified following the scheme depicted in    FIG. 7 .-   20. The composition of any one of clauses 1-19, wherein the spacer    sequence comprises an ANGPTL3 ABE guide RNA spacer sequence set    forth in Table 1.-   21. The composition of clause 20, wherein the guide RNA comprises    the ANGPTL3 ABE guide RNA sequence of GA067, GA091, GA098, GA099,    GA100, GA101, GA102, GA103, GA347, GA441, GA442, GA472, GA473,    GA474, GA475, GA476, GA517 or GA547 as set forth in Table 1.-   22. The composition of any one of clauses 1-19, wherein the    protospacer sequence comprises an ANGPTL3 ABE protospacer sequence    set forth in Table 1.-   23. The composition of clause 22, wherein the protospacer comprises    the sequence 5′-AAGATACCTGAATAACTCTC-3′ (SEQ ID No: 14),    5′-AAGATACCTGAATAACCCTC-3′ (SEQ ID No: 15), 5′-GATACCTGAATAACTCTC-3′    (SEQ ID No: 1606), 5′-AGATACCTGAATAACCCTC-3′ (SEQ ID No: 248), or    5′-GATACCTGAATAACCCTC-3′ (SEQ ID No: 249).-   24. The composition of any one of clauses 1-23, wherein the base    editor fusion protein comprises an amino acid sequence of SEQ ID No:    2137.-   25. The composition of any one of clauses 1-24, wherein the GC %    content of the mRNA sequence is greater than 50%.-   26. The composition of clause 25, wherein the GC % content of the    mRNA sequence is greater than 56%.-   27. The composition of clause 26, wherein the GC % content of the    mRNA sequence is greater than or equal to 63%.-   28. The composition of clause 25, wherein the mRNA comprises an    adenine tTNA deaminase (TadA) region, a Cas9 region and a nuclear    localization sequence (NLS) region.-   29. The composition of clause 28, wherein the mRNA further comprises    a first linker region which connects the TadA region and the Cas9    region, and a second linker region which connects the Cas9 region    and the NLS region.-   30. The composition of clause 28 or 29, wherein the GC % content of    the TadA region is greater than 60%.-   31. The composition of clause 28 or 29, wherein the GC % content of    the TadA region is greater than or equal to 70%.-   32. The composition of clause 28 or 29, wherein the GC % content of    the Cas9 region is greater than 56%.-   33. The composition of clause 28 or 29, wherein the GC % content of    the Cas9 region is greater than or equal to 62%.-   34. The composition of clause 28 or 29, wherein the GC % content of    the NLS region is greater than 54%.-   35. The composition of clause 28 or 29, wherein the GC % content of    the NLS region is greater than or equal to 63%.-   36. The composition of clause 29, wherein the GC % content of the    first linker region is greater than 65%.-   37. The composition of clause 29, wherein the GC % content of the    first linker region is greater than or equal to 79%.-   38. The composition of clause 29, wherein the GC % content of the    second linker region is greater than 67%.-   39. The composition of clause 29, wherein the GC % content of the    second linker region is greater than or equal to 83%.-   40. The composition of clause 29, wherein the GC % content of the    TadA region is greater than 60%, the GC % content of the Cas9 region    is greater than 56%, the GC % content of the NLS region is greater    than 54%, the GC % content of the first linker region is greater    than 65%, and the GC % content of the second linker region is    greater than 67%.-   41. The composition of clause 25, wherein the mRNA comprises a mRNA    sequence selected from Table 23.-   42. The composition of clause 41, wherein the mRNA comprises a mRNA    sequence of SEQ ID No: 2136.-   43. The composition of any one of clauses 25-42, wherein the mRNA    comprises a poly A tail.-   44. The composition of any one of clauses 1-43, further comprising a    lipid nanoparticle (LNP) enclosing (i).-   45. The composition of clause 44, wherein the LNP further encloses    (ii).-   46. The composition of clause 44, further comprising a second LNP    enclosing (ii).-   47. The composition of any one of clauses 1-44, wherein the ratio of    the guide RNA and the mRNA encoding the base editor fusion protein    is about 1:10 to about 10:1 by weight.-   48. The composition of clause 47, wherein the ratio of the guide RNA    and the mRNA encoding the base editor fusion protein is about 1:1,    1.5:1, 2:1, 3:1, 4:1, 1:1.5, 1:2, 1:3, or 1:4 by weight.-   49. The composition of clause 48, wherein the ratio of the guide RNA    and the mRNA encoding the base editor fusion protein is about 1:1 by    weight.-   50. A pharmaceutical composition comprising the composition of any    one of the preceding clauses and a pharmaceutically acceptable    carrier or excipient.-   51. A method for treating or preventing a condition in a subject in    need thereof, the method comprising administering to the subject a    therapeutically effective amount of the composition of clause 1.-   52. The method of clause 51, wherein the administration is via    intravenous infusion.-   53. The method of clause 51 or 52, comprising sequential    administration of a LNP enclosing (i) and a LNP enclosing (ii).-   54. The method of clause 51 or 52, comprising concurrent    administration of the LNP enclosing (i) and the LNP enclosing (ii).-   55. The method of clause 53, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 1 day.-   56. The method of clause 53, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 2 days.-   57. The method of clause 53, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 3 days.-   58. The method of clause 53, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 4 days.-   59. The method of clause 53, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 5 days.-   60. The method of clause 53, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 6 days.-   61. The method of clause 53, comprising administering a single dose    of the LNP enclosing (ii) followed by staggered doses of the LNP    enclosing (i) over an interval of 7 days.-   62. The method of clause 51 or 52, comprising administering a single    dose of the LNP enclosing (i) and (ii).-   63. The method of clause 62, wherein the single dose of the LNP is    at about 0.3 to about 3 mg/kg.-   64. The method of clause 62 or 63, comprising administering a    treatment course of one or more treatments to the subject, wherein    each one of the one or more treatment comprises one or more of the    single doses of the LNP.-   65. The method of clause 64, comprising administering a treatment    course of two to ten treatments.-   66. The method of clause 64, comprising administering a treatment    course of two to five treatments.-   67. The method of clause 64, comprising administering a treatment    course of two treatments.-   68. The method of clause 64, comprising administering a treatment    course of three treatments.-   69. The method of clause 64, comprising administering a treatment    course of four treatments.-   70. The method of clause 62, comprising administering a treatment    course of five treatments.-   71. The method of any one of clauses 51-70, wherein the condition is    an atherosclerotic cardiovascular disease.-   72. The method of any one of clauses 51-70, wherein the condition is    an atherosclerotic vascular disease.-   73. The method of any one of clauses 51-72, wherein the subject is a    human.-   74. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a ANGPTL3 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the ANGPTL3 gene in vivo when    administered to a mammalian subject, and-   wherein the guide RNA comprises the ANGPTL3 ABE guide RNA sequences    as set forth in Table 1.-   75. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a ANGPTL3 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the ANGPTL3 gene in vivo when    administered to a mammalian subject, and-   wherein the mRNA comprises a sequence selected from Table 23.-   76. A method for treating or preventing an atherosclerotic    cardiovascular disease in a subject in need thereof, the method    comprising administering to the subject a therapeutically effective    amount of a first composition, comprising-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a PCSK9 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the PCSK9 gene in vivo when    administered to a mammalian subject,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.5 mg/kg, the base alteration occurs in at least    35% of whole liver cells in the mammalian subject as measured by    next generation sequencing or Sanger sequencing; and-   a second composition, comprising-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a ANGPTL3 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the ANGPTL3 gene in vivo when    administered to a mammalian subject,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 1 mg/kg, the base alteration occurs in at least    35% of whole liver cells in the mammalian subject as measured by    next generation sequencing or Sanger sequencing.-   77. The method of clause 76, comprising sequential administration of    the first composition and the second composition.-   78. The method of clause 77, comprising administering one or more    doses of the first composition followed by one or more dose of the    second composition.-   79. The method of clause 78, comprising administering one or more    doses of the second composition followed by one or more dose of the    first composition.-   80. The method of clause 76, comprising concurrent administration of    the first composition and the second composition.-   81. The method of clause 80, comprising one or more doses of the    first composition and the second composition.-   82. A composition for editing a gene target comprising:-   (i) a base editor fusion protein comprising a programmable DNA    binding domain and a deaminase, or a mRNA encoding the same,-   (ii) a guide RNA comprising a tracr sequence that serves as a    binding scaffold for the base editor fusion protein, and a spacer    sequence that corresponds to a protospacer on a ANGPTL3 gene,-   wherein the guide RNA directs the base editor fusion protein to    effect a nucleobase alteration in the ANGPTL3 gene in vitro,-   wherein when the guide RNA and the mRNA is administered at a total    amount of at least 0.5 mg/kg, the base alteration occurs in at least    35% of whole liver cells in the mammalian subject as measured by    next generation sequencing or Sanger sequencing.-   83. The composition of clause 82, wherein when the guide RNA and the    mRNA is administered at a total amount of at least 1 mg/kg, the base    alteration occurs in at least 40% of whole liver cells in the    mammalian subject as measured by next generation sequencing or    Sanger sequencing.-   84. The composition of clause 82, wherein when the guide RNA and the    mRNA is administered at a total amount of at least 1.5 mg/kg, the    base alteration occurs in at least 45% of whole liver cells in the    mammalian subject as measured by next generation sequencing or    Sanger sequencing.-   85. The composition of clause 82, wherein when the guide RNA and the    mRNA is administered at a total amount of at least 2 mg/kg, the base    alteration occurs in at least-   50% of whole liver cells in the mammalian subject as measured by    next generation sequencing or Sanger sequencing.-   86. The composition of clause 82, wherein when the guide RNA and the    mRNA is administered at a total amount of at least 2.5 mg/kg, the    base alteration occurs in at least 55% of whole liver cells in the    mammalian subject as measured by next generation sequencing or    Sanger sequencing.-   87. The composition of clause 82, wherein when the guide RNA and the    mRNA is administered at a total amount of at least 3 mg/kg, the base    alteration occurs in at least 60% of whole liver cells in the    mammalian subject as measured by next generation sequencing or    Sanger sequencing.-   88. A composition for editing an ANGPTL3 gene comprising:    -   (a) a mRNA encoding an adenine base editor protein having an        editing window, and    -   (b) a guide RNA comprising a tracr sequence that serves as a        binding scaffold for the base editor protein and a spacer        sequence that serves to guide the base editor protein to a        protospacer sequence on the ANGPTL3 gene,-   wherein the spacer sequence is complimentary, at least in part, to a    splice site or an exon region of the ANGPTL3 gene.-   89. The composition for editing the ANGPTL3 gene of clause 83,    wherein when the base editor protein is operatively bound to the    guide RNA and the guide RNA is hybridized with the complementary    strand to the protospacer sequence on the ANGPTL3 gene, the editing    window encompasses the splice site of the ANGPTL3 gene.-   90. The composition for editing the ANGPTL3 gene of clause 88,    wherein when the base editor protein is operatively bound to the    guide RNA and the guide RNA is hybridized with the complementary    strand to the protospacer sequence on the ANGPTL3 gene, the editing    window encompasses a region of an intron of the ANGPTL3 gene.-   91. The composition for editing the ANGPTL3 gene of clause 88,    wherein when the base editor protein is operatively bound to the    guide RNA and the guide RNA is hybridized with the complementary    strand to the protospacer sequence on the ANGPTL3 gene, the editing    window encompasses a region of intron 1, intron 3 or intron 4 of the    ANGPTL3 gene.-   92. The composition for editing the ANGPTL3 gene of clause 88,    wherein when the base editor protein is operatively bound to the    guide RNA and the guide RNA is hybridized with the complementary    strand to the protospacer sequence on the ANGPTL3 gene, the editing    window encompasses a region of intron 1 of the ANGPTL3 gene.-   93. The composition for editing the ANGPTL3 gene of clause 88,    wherein the spacer sequence has a 80-100% nucleotide sequence    identity to a spacer sequence selected from the group of guide RNA    sequences identified as GA067, GA100 and GA574.-   94. The composition for editing the ANGPTL3 gene of clause 88,    wherein the tracr sequence has a 80-100% nucleotide sequence    identity to a tracr sequence selected from the group of guide RNA    sequences identified as GA067, GA091, GA098, GA099, GA100, GA101,    GA102, GA103, GA347, GA441, GA442, GA472, GA473, GA474, GA475,    GA476, GA517 and GA547.-   95. The composition for editing the ANGPTL3 gene of clause 88,    wherein the mRNA has an 80-100% sequence identity to the mRNA    sequences identified as MA002, MA004, MA040, MA0041, or MA045.-   96. The composition for editing the ANGPTL3 gene of clause 88,    wherein the mRNA has one or more of the GC nucleotide region    percentages set forth in the following table:

Nucleotide region Average GC Nucleotide Content  27-213 67-73% 389-66167-71% 735-829 63-74% 4207-4286 67-70% 4537-4569 65-73% 4683-4741 62-67%

-   97. The composition for editing the ANGPTL3 gene of clause 88,    wherein the mRNA has one or more of the GC nucleotide region    percentages set forth in the following table:

Nucleotide region Average GC Nucleotide Content  27-213 At least 73%389-661 At least 71% 735-829 At least 74% 4207-4286 At least 70%4537-4569 At least 73% 4683-4741 At least 67%

-   98. The composition for editing the ANGPTL3 gene of clause 88,    wherein the mRNA and gRNA are encapsulated within a lipid    nanoparticle.-   99. The composition for editing the ANGPTL3 gene of clause 88,    wherein the mRNA and gRNA are encapsulated within a lipid    nanoparticle having the following:-   LNP composition (mol %):-   40-65% iLipid-   2-20% DSPC-   1-5% PEG-   Remaining mol % balance is cholesterol;-   LNP Particle size: 55-120 nm Z average hydrodynamic diameter; and-   Polydispersity index of <0.2 as determined by dynamic light    scattering.-   100. The composition for editing the ANGPTL3 gene of clause 88,    wherein the mRNA and gRNA are encapsulated within the lipid    nanoparticle having an LNP particle size between 50-70 nm Z average    hydrodynamic diameter.-   101. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   102. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   103. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 30    percent.-   104. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   105. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   106. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 60    percent.-   107. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 70    percent.-   108. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   109. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   110. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 60    percent.-   111. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 70    percent.-   112. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when at administered to a group of    cynomolgus monkeys via intravenous infusion at a dose of    approximately 1.5 mg of the guide RNA and mRNA combined total weight    per kg of the cynomolgus monkey weight is capable of inducing    adenine base editing at the ANGPTL3 target splice site in the liver    of the cynomolgus monkeys with an average editing percentage of    greater than 80 percent.-   113. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 40    percent.-   114. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 50    percent.-   115. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 60    percent.-   116. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 70    percent.-   117. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of the    cynomolgus monkey weight is capable of inducing adenine base editing    at the ANGPTL3 target splice site in the liver of the cynomolgus    monkeys with an average editing percentage of greater than 80    percent.-   118. The compositions for editing the ANGPTL3 gene of clauses    87-117, wherein the percent editing is determined at 15 days after    dosing through analysis of dosed cynomolgus monkey liver either via    liver biopsy or necropsy of the monkey.-   119. The compositions for editing of the ANGPTL3 gene of clauses    87-117, wherein the percent editing is determined to be durably    maintained by periodic liver biopsy testing of the dosed cynomolgus    monkeys over a span of at least 168 days after dosing.-   120. The compositions for editing of the ANGPTL3 gene of clauses    87-117, wherein the percent editing is determined to be durably    maintained by periodic liver biopsy testing of the dosed cynomolgus    monkeys over a span of at least 300 days after dosing.-   121. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   122. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   123. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of monkey    weight is capable of reducing ANGPTL3 protein in the plasma of the    dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   124. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed monkeys on average of at least 60 percent as compared    to baseline.-   125. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   126. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   127. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   128. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   129. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of monkey weight    is capable of reducing ANGPTL3 protein in the plasma of the dosed    cynomolgus monkeys on average of at least 50 percent as compared to    baseline.-   130. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   131. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   132. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   133. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   134. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   135. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   136. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   137. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   138. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   139. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 35 percent as    compared to baseline.-   140. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 40 percent as    compared to baseline.-   141. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 50 percent as    compared to baseline.-   142. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 60 percent as    compared to baseline.-   143. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 70 percent as    compared to baseline.-   144. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing ANGPTL3 protein in the plasma    of the dosed cynomolgus monkeys on average of at least 80 percent as    compared to baseline.-   145. The compositions for editing of the ANGPTL3 gene of clauses    121-144, wherein the reduction in plasma protein is determined at 15    days after dosing via blood sampling and analysis of the dosed    cynomolgus monkey.-   146. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of monkey    weight is capable of reducing triglyceride level in the plasma of    the dosed monkeys on average of at least 20 percent as compared to    baseline.-   147. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 25    percent as compared to baseline.-   148. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 30    percent as compared to baseline.-   149. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 35    percent as compared to baseline.-   150. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 40    percent as compared to baseline.-   151. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 0.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 45    percent as compared to baseline.-   152. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 20    percent as compared to baseline.-   153. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 25    percent as compared to baseline.-   154. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 30    percent as compared to baseline.-   155. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 35    percent as compared to baseline.-   156. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 40    percent as compared to baseline.-   157. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 45    percent as compared to baseline.-   158. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 50    percent as compared to baseline.-   159. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 55    percent as compared to baseline.-   160. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 60    percent as compared to baseline.-   161. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 20    percent as compared to baseline.-   162. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 25    percent as compared to baseline.-   163. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 30    percent as compared to baseline.-   164. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 35    percent as compared to baseline.-   165. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 40    percent as compared to baseline.-   166. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 45    percent as compared to baseline.-   167. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 50    percent as compared to baseline.-   168. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 55    percent as compared to baseline.-   169. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 60    percent as compared to baseline.-   170. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 1.5 mg    of the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 65    percent as compared to baseline.-   171. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 20    percent as compared to baseline.-   172. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 25    percent as compared to baseline.-   173. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 30    percent as compared to baseline.-   174. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 35    percent as compared to baseline.-   175. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 40    percent as compared to baseline.-   176. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 45    percent as compared to baseline.-   177. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 50    percent as compared to baseline.-   178. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 55    percent as compared to baseline.-   179. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 60    percent as compared to baseline.-   180. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing triglyceride level in the    plasma of the dosed cynomolgus monkeys on average of at least 65    percent as compared to baseline.-   181. The compositions for editing the ANGPTL3 gene of clauses    148-182, wherein the reduction in triglyceride level is determined    at 15 days after dosing via blood sampling and analysis of the dosed    cynomolgus monkey.-   182. The compositions for editing the ANGPTL3 gene of clauses    148-182, wherein the reduction in triglyceride level is determined    to be durably maintained over a span of at least 168 days by    periodic blood sampling and analysis of the dosed cynomolgus monkey.-   183. The compositions for editing the ANGPTL3 gene of clauses    148-182, wherein the reduction in triglyceride level is determined    to be durably maintained over a span of at least 300 days by    periodic blood sampling and analysis of the dosed cynomolgus monkey.-   184. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) in the plasma of    the dosed cynomolgus monkeys on average of at least 10 percent as    compared to baseline.-   185. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 15    percent as compared to baseline.-   186. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 20    percent as compared to baseline.-   187. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 25    percent as compared to baseline.-   188. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of at least 30    percent as compared to baseline.-   189. The composition for editing the ANGPTL3 gene of clause 88,    wherein the composition when administered to a group of cynomolgus    monkeys via intravenous infusion at a dose of approximately 3 mg of    the guide RNA and mRNA combined total weight per kg of cynomolgus    monkey weight is capable of reducing lipoprotein(a) level in the    plasma of the dosed cynomolgus monkeys on average of approximately    35 percent as compared to baseline.-   190. The compositions for editing the ANGPTL3 gene of clauses    186-191, wherein the reduction in lipoprotein(a) is determined at 15    days after dosing via blood sampling and analysis of the dosed    cynomolgus monkey.-   191. The compositions for editing the ANGPTL3 gene of clauses    186-191, wherein the reduction in lipoprotein(a) is determined to be    durably maintained over a span of at least 224 days by periodic    blood sampling and analysis of the dosed cynomolgus monkey.-   192. The compositions for editing the ANGPTL3 gene of clauses    186-191, wherein the reduction in lipoprotein(a) is determined to be    durably maintained over a span of at least 300 days by periodic    blood sampling and analysis of the dosed cynomolgus monkey.-   193. The compositions for editing the ANGPTL3 gene of clauses    101-194, wherein to the extent that the dosing of the cynomolgus    monkeys results in elevation of AST, ALT, or Cytokines, the    elevations resulting from the dosing of the composition are    transient and resolved back to approximately baseline levels within    3-15 days after dosing.-   194. The composition for editing the ANGPTL3 gene of clauses    101-103, 121-126, 148-153, wherein the percent editing of ANGPTL3 is    negligible outside of the liver, spleen and adrenal glands tissues    as illustrated in FIG. 27 .-   195. The compositions for editing the ANGPTL3 gene of clauses    101-107, 121-132, 148-162, wherein repeat dosing results is additive    with respect to the editing percentage of ANGPTL3 editing    percentage.-   196. The compositions for editing the ANGPTL3 gene of clause 195,    wherein the repeat dosing does not elicit cytokine activation nor an    immune response.-   197. The composition for editing the ANGPTL3 gene of clause 88,    wherein the spacer sequence has at least 80% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the    ANGPTL3 gene, wherein an RNA nucleotide on the spacer sequence is in    correlation with a DNA nucleotide of the protospacer if it has the    same nucleotide as the DNA nucleotide in the same order and wherein    uracil and thymine bases are considered the same nucleotide for    purposes of determining correlation.-   198. The composition for editing the ANGPTL3 gene of clause 197,    wherein the spacer sequence has at least 85% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the    ANGPTL3 gene.-   199. The composition for editing the ANGPTL3 gene of clause 197,    wherein the spacer sequence has at least 90% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the    ANGPTL3 gene.-   200. The composition for editing the ANGPTL3 gene of clause 197,    wherein the spacer sequence has at least 95% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the    ANGPTL3 gene.-   201. The composition for editing the ANGPTL3 gene of clause 197,    wherein the spacer sequence has at least 99% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the    ANGPTL3 gene.-   202. The composition for editing the ANGPTL3 gene of clause 197,    wherein the spacer sequence has at least 100% nucleotide correlation    with the nucleotide sequence of a targeted protospacer on the    ANGPTL3 gene.-   203. A method for treating or preventing an atherosclerotic    cardiovascular disease in a subject in need thereof, the method    comprising administering to the subject a therapeutically effective    amount of    -   (a) a mRNA encoding an adenine base editor protein having an        editing window,    -   (b) a first guide RNA comprising a tracr sequence that serves as        a binding scaffold for the base editor protein and a spacer        sequence that serves to guide the base editor protein to a        protospacer sequence on a PCSK9 gene, wherein the spacer        sequence is complimentary, at least in part, to a splice site or        an exon region of the PCSK9 gene; and    -   (c) a second guide RNA comprising a tracr sequence that serves        as a binding scaffold for the base editor protein and a spacer        sequence that serves to guide the base editor protein to a        protospacer sequence on a ANGPTL3 gene, wherein the spacer        sequence is complimentary, at least in part, to a splice site or        an exon region of the ANGPTL3 gene.-   204. The method of clause 203, further comprising a first LNP    enclosing (a).-   205. The method of clause 204, wherein the first LNP encloses (b)    and (c).-   206. The method of clause 204, wherein the first LNP was    administered repeatedly.-   207. The method of clause 204, wherein the first LNP was    administered repeatedly at an interval of one to sixty days.-   208. The method of clause 204, wherein the first LNP was    administered repeatedly at an interval of seven days.-   209. The method of clause 204, wherein the first LNP further    encloses (b).-   210. The method of clause 209, further comprising a second LNP    enclosing (a) and (c).-   211. The method of clause 210, wherein the first LNP and the second    LNP are administered sequentially.-   212. The method of clause 211, wherein the first LNP and the second    LNP are administered sequentially at an interval of one day to 12    months.-   213. The method of clause 212, wherein the interval is one day.-   214. The method of clause 212, wherein the interval is five days.-   215. The method of clause 212, wherein the interval is ten days.-   216. The method of clause 212, wherein the interval is fifteen days.-   217. The method of clause 212, wherein the interval is twenty days.-   218. The method of clause 212, wherein the interval is twenty-five    days.-   219. The method of clause 212, wherein the interval is one month.-   220. The method of clause 212, wherein the interval is two months.-   221. The method of clause 212, wherein the interval is three months.-   222. The method of clause 212, wherein the interval is five months.-   223. The method of clause 212, wherein the interval is eight months.-   224. The method of clause 212, wherein the interval is ten months.-   225. The method of clause 212, wherein the interval is twelve    months.

It will also be appreciated from reviewing the present disclosure, thatit is contemplated that the one or more aspects or features presented inone of or a group of related clauses may also be included in otherclauses or in combination with the one or more aspects or features inother clauses.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20230158174A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A single guide RNA that comprises: (a) a spacersequence, wherein the spacer sequence comprises (i) one or more chemicalmodification(s) and (ii) one or more unmodified nucleotide(s) at selectposition(s), and (b) a tracr sequence having at least 70% identity toSEQ ID NO: 61, wherein the tracr sequence serve as a binding scaffoldfor a Type II Cas protein, and wherein the tracr sequence comprises (i)one or more chemical modification(s) and (ii) one or more unmodifiednucleotide(s) at select position(s).
 2. The single guide RNA of claim 1,wherein the spacer sequence hybridizes with a target polynucleotidesequence in a gene of interest when contacted with the targetpolynucleotide sequence, wherein the single guide RNA directs the Casprotein to effect alteration in the gene when administered to amammalian subject.
 3. The single guide RNA of claim 1 or 2, wherein thetracr sequence comprises unmodified nucleotides at positions 2 to 7, 23to 25, 27, 29, 31, 38, 39, 42 to 45 48, 49 and 62 as numbered in SEQ IDNO: 61 or a corresponding position thereof.
 4. The single guide RNA ofclaim 1 or 2, wherein the tracr sequence comprises unmodifiednucleotides at positions 2 to 7, 13, 23 to 25, 27, 29, 31, 38, 39, 42 to45, 48, 49, 53 and 62 as numbered in SEQ ID NO: 61 or a correspondingposition thereof.
 5. The single guide RNA of claim 1 or 2, wherein thetracr sequence comprises unmodified nucleotides at positions 2 to 7, 13,23 to 25, 27, 29, 31, 38, 39, 42 to 45, 48, 49, 53, 61, 68, 70 and 11 asnumbered in SEQ ID NO: 61 or a corresponding position thereof.
 6. Thesingle guide RNA of any one of claims 1 to 3, wherein the tracr sequencecomprises modified nucleotides at positions 1, 8 to 22, 26, 28, 30, 32to 37, 40, 41, 46, 47, 50 to 61, and 63 to 80 as numbered in SEQ ID NO:61 or a corresponding position thereof.
 7. The single guide RNA of anyone of claims 1 to 3, wherein the tracr sequence comprises modifiednucleotides at positions 1, 8 to 12, 14 to 22, 26, 28, 30, 32 to 37, 40,41, 46, 47, 50 to 52, 54 to 61, and 63 to 80 as numbered in SEQ ID NO:61 or a corresponding position thereof.
 8. The single guide RNA of anyone of claims 1 to 7, wherein the tracr sequence comprises modifiednucleotides at positions 1, 8 to 12, 14 to 22, 26, 28, 30, 32 to 34, 37,41, 46, 47, 50 to 52, 54 to 60, 62 to 67, 69 and 72 to 80 as numbered inSEQ ID NO: 61 or a corresponding position thereof.
 9. The single guideRNA of any one of claims 3 to 8, wherein more than 60% of thenucleotides in the tracr sequence with SEQ ID NO: 61 are modified. 10.The single guide RNA of claim 9, wherein more than 70% of thenucleotides in the tracr sequence with SEQ ID NO: 61 are modified. 11.The single guide RNA of any one of claims 1 to 10, wherein the tracrsequence comprises unmodified nucleotides at positions 13, 49, 53 and 62as numbered in SEQ ID NO: 61 or a corresponding position thereof. 12.The single guide RNA of any one of claim 11, wherein the tracr sequencecomprises unmodified nucleotides at positions 49 and 62 as numbered inSEQ ID NO: 61 or a corresponding position thereof.
 13. The single guideRNA of any one of claims 1 to 10, wherein the tracr sequence comprisesunmodified nucleotides at positions 13, 40, 49, 53, 61, 68, 70 and 71 asnumbered in SEQ ID NO: 61 or a corresponding position thereof.
 14. Thesingle guide RNA of any one of claim 13, wherein the tracr sequencecomprises unmodified nucleotides at positions 13, 49 and 53 as numberedin SEQ ID NO: 61 or a corresponding position thereof.
 15. The singleguide RNA of any one of claims 1 to 10, wherein the tracr sequencecomprises modified nucleotides at positions 1, 8, 21, 22, 26, 28, 30, 32to 37, 40, 41, 46 and 47 as numbered in SEQ ID NO: 61 or a correspondingposition thereof.
 16. The single guide RNA of any one of claims 1-15,wherein the tracr sequence comprises at least 80% identity to SEQ ID NO:61.
 17. The single guide RNA of any one of claims 1-15, wherein thetracr sequence comprises at least 90% identity to SEQ ID NO:
 61. 18. Thesingle guide RNA of any one of claims 1-17, wherein the chemicalmodification comprises a 2′-OMe modification.
 19. The single guide RNAof any one of claims 1-17, wherein the chemical modification comprises anebularin or a deoxynebularin.
 20. The single guide RNA of any one ofclaims 1-17, wherein the chemical modification comprises aphosphorothioate linkage.
 21. The single guide RNA of any one of claims1-20, wherein the tracr sequence comprises SEQ ID No. 61, and whereinthe single guide RNA further comprises one or more phosphorothioatelinkage at a 5′ end, at a 3′ end, at select internal positions or anycombinations thereof.
 22. The single guide RNA of claim 21, wherein thesingle guide RNA further comprises two and no more than two contiguousphosphorothioate linkages at the 5′ end, at the 3′ end or both.
 23. Thesingle guide RNA of claim 21, wherein the single guide RNA furthercomprises three contiguous phosphorothioate linkages at the 5′ end, atthe 3′ end or both.
 24. The single guide RNA of any one of claims 1-21,wherein the single guide RNA comprises the sequence 5′- ususuNNN-3′ atthe 3′end, wherein N independently indicates a unmodifiedribonucleotide, and wherein each u indicates 2′-O-methyluridine and eachs indicates phosphorothioate linkage.
 25. The single guide RNA of claim24, wherein each N is uridine.
 26. The single guide RNA of any one ofclaims 1-21, wherein the single guide RNA comprises the sequence 5′-ususuNNn-3′ at the 3′end, wherein each N independently indicates aunmodified ribonucleotide, wherein the n indicates a modifiednucleotide, wherein each u indicates 2′-O-methyluridine and wherein eachs indicates phosphorothioate linkage.
 27. The single guide RNA of claim26, wherein each N is uridine and the n is 2′-O-methyluridine.
 28. Asingle guide RNA that comprises (i) a spacer sequence and (ii) a tracrsequence, wherein the spacer sequence hybridizes with a targetpolynucleotide sequence in a PCSK9 gene or an ANGPTL3 gene whencontacted with the target polynucleotide sequence, wherein the tracrsequence binds a Type II Cas protein when contacted with the Type II Casprotein, and wherein the single guide RNA comprises a nebularine, adeoxynebularine, or a 2′-O-methylnebularine.
 29. A single guide RNA thatcomprises (i) a spacer sequence and (ii) a tracr sequence, wherein thespacer sequence hybridizes with a target polynucleotide sequence in aPCSK9 gene or an ANGPTL3 gene when contacted with the targetpolynucleotide sequence, wherein the tracr sequence binds a Type II Casprotein when contacted with the Type II Cas protein, and wherein thesingle guide RNA comprises two and no more than two phosphorothioatelinkages at a 5′ end or at a 3′ end.
 30. The single guide RNA of claim29, wherein the single guide RNA comprises two and no more than twophosphorothioate linkages at the 5′ end and at the 3′ end.
 31. Thesingle guide RNA of claim 29, wherein the single guide RNA comprisesthree phosphorothioate linkages at the 5′ end.
 32. The single guide RNAof claim 29, wherein the single guide RNA comprises threephosphorothioate linkages at the 3′ end.
 33. The single guide RNA ofclaim 29, wherein the two phosphorothioate linkages at the 5′ end aretwo contiguous phosphorothioate linkages at the first two nucleotidepositions of the 5′ end.
 34. The single guide RNA of claim 29, whereinthe two phosphorothioate linkages at the 5′ end are within the first3-10 nucleotides of the 5′ end.
 35. The single guide RNA of claim 29,wherein the two phosphorothioate linkages at the 3′ end are twocontiguous phosphorothioate linkages at the last two nucleotidepositions of the 3′ end.
 36. The single guide RNA of claim 29, whereinthe two phosphorothioate linkages at the 3′ end are within the last 3-10nucleotides of the 3′ end.
 37. The single guide RNA of claim 29comprising the sequence 5′-UsUsUs-3′ at the 3′ end, wherein U indicatesa uridine and s indicates a phosphorothioate linkage.
 38. The singleguide RNA of claim 29 comprising the sequence 5′-UUU-3′ at the 3′end.39. The single guide RNA of any one of claims 1-27, wherein the tracrsequence binds the Type II Cas protein with increased binding affinitycompared to a tracr sequence in an unmodified single guide RNA.
 40. Thesingle guide RNA of any one of claims 28-38, wherein the Type II Casprotein is a Cas9 protein.
 41. The single guide RNA of claim 40, whereinthe Cas9 protein is a Streptococcus pyogenes Cas9.
 42. A single guideRNA that comprises a guide RNA sequence selected from Table 1, whereina, u, g, and c indicate 2′-OMe modified adenine, uridine, guanine, andcytidine, wherein s indicates a phosphorothioate linkage, wherein Xindicates a nebularine, wherein x indicates a 2′-O-methylnebularine andwherein dX indicates a 2′-deoxynebularine.
 43. A pharmaceuticalcomposition for gene modification comprising the single guide RNA of anyone of claims 1-42 and a Type II Cas protein or a nucleic acid sequenceencoding the Type II Cas protein.
 44. The pharmaceutical composition ofclaim 43 further comprising a vector that comprises the nucleic acidsequence encoding the Type II Cas protein.
 45. The pharmaceuticalcomposition of claim 43 or 44, wherein the Type II Cas protein is aCas9.
 46. The pharmaceutical composition of any one of claims 43-45further comprising a pharmaceutically acceptable carrier.
 47. A lipidnanoparticle comprising the pharmaceutical composition of any one ofclaims 43-46.
 48. A method for modifying a target polynucleotidesequence in a cell comprising introducing into the cell thepharmaceutical composition of any one of claims 43-46, wherein thesingle guide RNA directs the Type II Cas protein to effect amodification in the target polynucleotide sequence in the cell.
 49. Themethod of claim 48, wherein the target polynucleotide sequence is in aPCSK9 gene.
 50. The method of claim 49, wherein the modification reducesexpression of functional PCSK9 protein encoded by the PCSK9 gene in thecell.
 51. The method of claim 50, wherein the target polynucleotidesequence is in an ANGPTL3 gene.
 52. The method of claim 51 wherein themodification reduces expression of functional ANGPTL3 protein encoded bythe ANGPTL3 gene in the cell.
 53. The method of any one of claims 48-52,wherein the introduction is performed via a lipid nanoparticle thatcomprises the composition.
 54. A method for treating or preventing acondition in a subject in need thereof, the method comprisingadministering to the subject the pharmaceutical composition of any oneof claims 43-46 or the lipid nanoparticle of claim 47, wherein thesingle guide RNA directs the Type II Cas protein to effect amodification in a target polynucleotide sequence in a cell of thesubject, thereby treating or preventing the condition.
 55. The method ofclaim 54, wherein the target polynucleotide sequence is in a PCSK9 gene.56. The method of claim 54, wherein the target polynucleotide sequenceis in an ANGPTL3 gene.
 57. The method of claim 55, wherein themodification reduces expression of functional PCSK9 protein encoded bythe PCSK9 gene in the subject.
 58. The method of claim 56, wherein themodification reduces expression of functional ANGPTL3 protein encoded bythe ANGPTL3 gene in the subject.
 59. The method of any one of claims48-58 wherein the condition is an atherosclerotic vascular disease. 60.The method of any one of claims 48-58, wherein the condition is anatherosclerotic vascular disease, hypertriglyceridemia, or diabetes. 61.The method of any one of claims 48-60, wherein the subject exhibits areduced blood LDL cholesterol level, and/or a reduced bloodtriglycerides level as compared to before the administration.